Cell, Vol. 66, 237-255,
January
24, 1992, Copyright
0 1992 by Cell Press
Mechanisms of Asymmetric Cell Division: Two Bs or Not Two Bs, That Is the Question H. Robert Horvitz” and Ira Herskowitzt *Howard Hughes Medical Institute Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 tDepartment of Biochemistry and Biophysics University of California, San Francisco San Francisco. California 94143
How sister cells can have different fates is a fundamental aspect of the problem of how cell diversity is generated during development. In principle, sister cells that aredifferent could be produced in two distinct ways. First, polar mother cells could divide to generate daughters that are different from the time they are formed. Alternatively, two identical sister cells could be generated and become different as a consequence of some later event. Consideration of these mechanisms raises immediate further questions: What causes a mother cell to be polar? How do initially identical sister cells become different? And in each case, how do initial differences in sister cells lead to their ultimately distinct fates? Answers to these questions would provide important insights into the mechanisms responsible for the control of development. In this review, we use the term “asymmetric cell division” to refer to any cell division in which sister cells have different fates. These distinct fates can be recognized by differences in size, shape, or in other morphological or biochemical features, or, if the sister cells are blast cells, by differences in their subsequent patterns of cell divisions and in the number and nature of the descendant cells they produce. Thus, if a cell division generates sister cells with distinct fates “A” and “B,” that division is considered to be asymmetric (Figure 1). By contrast, if a division generates sister cells with the same fate, e.g., 8, that division is symmetric. In short, the problem of asymmetric cell division is how a cell can divide to produce cells with distinct fates A and B instead of two cells that express the same fate B. As we use the term, an asymmetric cell division need not produce sister cells of different sizes and can produce initially identical cells with fates tllat differ as a consequence of subsequent cell interactions. Our current understanding of the mechanisms responsible for asymmetric cell division derives from a wide variety of experimental approaches using many different organisms. For example, classical observations of animal embryology established that specific regions of egg cytoplasm are partitioned to specific descendant cells that have distinct fates and also that certain substances are localized within the cytoplasm of the egg and can be differentially distributed to daughter cells. More recent studies involving micromanipulation or using a microelectrode to kill specific cells have revealed that cell interactions can cause sister cells to express different fates. Genes that
Review
are functionally responsible for asymmetric cell division have been identified in genetically tractable animals, such as nematodes and fruit flies, and the characterization of these genes has revealed aspects of the mechanisms by which they act. The most detailed studies of the mechanisms of asymmetric cell division have used easily studied unicellular organisms, including bacteria, yeast, and algae. Together these approaches are revealing the mechanisms responsible for specific asymmetric cell divisions and the degree to which these mechanisms are shared among organisms. We describe in this review examples of the two basic mechanisms noted above by which sister cells can become different, focusing on experimental systems in which the process can be studied at the level of single cells. We consider genetic analyses that have identified mutants in which the asymmetry of specific cell divisions is disrupted and what is known about the molecular mechanisms of action of the genes defined by these mutants. Finally, we discuss the similarities and differences among the mechanisms of asymmetric cell division that have been revealed by these various studies. Intrinsically
Asymmetric
Cell Divisions
The hypothesis that a cell division can produce two daughters that are intrinsically different from the time they are generated was formulated more than a century ago based upon studies of leech cell lineage, which revealed that distinct cytoplasmic domains of the leech egg are differentially partitioned to its descendants (Whitman, 1878). Subsequent observations of ascidian cell lineage supported this idea by identifying five distinct pigmented areas of egg cytoplasm that segregate to cells generating five distinct tissue types (Conklin, 1905). Such classical findings have been complemented by many modern studies in which specific molecules have been identified that are localized within specific regions of eggs and early blastomeres and that are distributed to specific descendant cells. Collectively, these observations have provided the basis for a fundamsntal hypothesis of developmental biology: specific molecules localized within regions of the cytoplasm of a mother cell can be distributed unequally to its two daughter cells and can act as developmental determinants to cause these daughters to express distinct characteristics and have different fates (Figure 2a). A comprehensive and stimulating review of the concept of cytoplasmic localization has been published by Davidson (1988). Mechanisms responsible for cytoplasmic localization of developmentally important molecules seem likely to be revealed by studies of Drosophila melanogaster genes that have RNA and/or protein products localized within tht egg (Driever and Niisslein-Volhard, 1988; Kim-Ha et al., 1991; Ephrussi et al., 1991; St Johnston et al., 1991). Although cytoplasmic localization provides an appealing mechanistic basis for intrinsically determined asymmetric cell division, other mechanisms can be imagined.
Cell 238
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DIVERSITY
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1. Generation
of Diversity
by Asymmetric
(a) Asymmetric cell division generates (see text), labeled A and f3. (b) Symmetric cell division generates
L
LOCALIZED DETERMINANTS
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ASYMMETRIC DIVISION PLANE
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Cell Division
sister cells of two distinct
SIGNALING
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sister cells of the same type, B.
Any molecule that is asymmetrically segregated could in principle be used to distinguish sister cells and hence to serve as a developmental determinant. Molecules localized to the cell cortex (as opposed to regions of the cytoplasm), to one of the two centrioles, or to one of the two DNA strands of a chromosome, or even those DNA strands themselves all are candidates of this sort. Furthermore, intrinsic asymmetry need not be caused by an asymmetrically segregated molecule. For example, the asymmetric placement of the mitotic spindle would result in the generation of daughter cells of different sizes, which could in turn cause the expression of different fates (Figure 2b). Evidence for a number of these mechanisms exists, as discussed below. Rigorous proof that a specific asymmetric cell division is intrinsically determined can be difficult to obtain and in many cases necessitates an understanding of the underlying mechanism. Even two sister cells that have been generated in isolation from other cells could become different as a consequence of signaling that occurs between the sisters. The existence of an asymmetrically segregated molecule or a difference in cell size is not sufficient to establish that differences in sister cell fates are intrinsically determined. Such differences must be shown to cause the sister cells to be different. For these reasons, many examples of apparent intrinsic determination rely on circumstantial evidence, such as the polarity of the mother cell, the inequality of the size of the daughter cells, or the asymmetric segregation of specific molecules. We discuss below some selected examples in which the intrinsic nature of the determination of an asymmetric cell division seems likely and in which the mechanistic basis of this asymmetry could well be revealed in the near future. Caulobacter The bacterium Caulobacter crescentus can exist as either of two distinct differentiated cell types (reviewed by Newton and Ohta, 1990; Gober and Shapiro, 1991). Swarmer cells are motile, contain elaborate flagella, and are chemo-
0
WITH OTHER CELLS
Figure
2. Mechanisms
of Asymmetric
Cell Division
(a and b) Intrinsic mechanisms. (a) Localized developmental determinants responsible for fates A and B are differentially segregated to the two daughter cells. (b) Asymmetry in the division plane results in daughters of different sizes, which can cause them to express the different fates A and B. (c and d) Mechanisms dependent upon cell signaling. Initially identical sister cells of bipotential fate AIB interact with each other or with other cells to become different and express fates A or B. Such cells are said to constitute sister cell equivalence groups (see text). (c) Initially identical sister cells become different as a consequence of interactions (indicated by the dashed line) with another cell or cells (indicated by the square). (d) Initially identical sister cells at random become biased toward one fate or the other and then reinforce that bias via interactions (dashed line) between the sister cells.
tactic. Stalked cells are nonmotile and can attach to the substratum through a stalk. Swarmer and stalked cells differ in many ways, including in their patterns of gene and protein expression. In addition, stalked cells can initiate DNA replication and divide, whereas swarmer cells can do so only after losing their flagella and becoming stalked cells. Stalked cells divide to produce one stalked cell and one swarmer cell (Figure 3a). What determines the biochemical and morphological differences between the sister cells produced by this asymmetric cell division? The swarmer cell contains a variety of swarmer cell-specific proteins, including the chemotaxis receptor encoded by the mcpA gene, the flagellin encoded by the flgK gene, and the hook protein encoded by the flaK gene (Gomes and Shapiro, 1984; Ohta et al., 1985; Minnich and Newton, 1987). Both these proteins and the mRNAs that encode them are prin-
Review: 239
Mechanisms
of Asymmetric
(a)
Cell Division
SWARMER
-0 STALKED
(b)
Figure
CELL
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DIFFERENT TRANSCRlPTlON
(c) pleC
CELL
FACTORS
DIFFERENT CHROMOSOMES
mutants
3. Caulobacter
(a) Only the nonmotile, stalked cell form of the wild-type bacterium Caulobacter crescentus can undergo cell division (Newton and Ohta, 1990; Gober and Shapiro, 1991). Within the predivisional cell, DNA replication occurs, and a flagellum is formed at the pole opposite to that at which the stalk is located. One daughter cell, like its mother, is a stalked cell, while the other is a motile and chemotactic swarmer cell. The swarmer cell can lose its flagellum, form a stalk, and then initiate cell division. (b) Models for how the pre-stalked and the pre-swarmer cell compartments of the predivisional cell differ in gene expression (see text). One possibility, shown on the left, is that these two compartments differ in their transcription factors (closed and open circles). Distinct proteins could be present or the same proteins could be differently modified causing them to be differentially active. A second possibility, shown on the right, is that the chromosomes in these two compartments are differentially active, perhaps as a consequence of the more condensed nature of the chromosome in the pre-swarmer cell compartment. (c) Mutants defective in the p/eC gene undergo a symmetric cell division, producing two progeny with defective flagella.
cipally synthesized in the predivisional cell (Gomes and Shapiro, 1984; Ohta et al., 1985), in which the flagellar apparatus assembles prior to cell division. After cell division, the swarmer cell continues to produce the f/gKflagellin (Minnich and Newton, 1987) apparently from mRNA synthesized in the predivisional cell and subsequently segregated to only the swarmer daughter cell (Milhausen and Agabian, 1983; Goberet al., 1991).Thisnewlysynthesized flagellin allows the flagellar filament to continue to grow.
These observations raise a number of questions concerning the asymmetric division that generates the swarmer and stalked cells. For example, what causes swarmer cellspecific proteins to be localized to the incipient swarmer cell pole of the predivisional cell? The answer, at least in part, is that some of these proteins, including the f/gKencoded flagellin, appear to contain sequences within them responsible for this localization (see Gober and Shapiro, 1991;Alleyet al., 1991). Theseproteinsmight associate with a site at the incipient swarmer cell pole (possibly a remnant from a prior cell division) that provides a morphogenetic landmark for assembling the complex flagellar and basal body structure. A second question also must be answered: what causes mRNA synthesized in the predivisional cell to be segregated specifically to the swarmer cell daughter? Localized transcription and the prevention of free diffusion within the predivisional cell seem to be responsible. Specifically, the late predivisional cell appears to consist of two compartments between which proteins and presumably other macromolecules do not freely exchange (Gober et al., 1991). Each of these compartments contains one of the two sister chromosomes present after replication but before division. Thus, if only one of these chromosomes expresses a particular complement of genes, the mRNA and protein products of those genes would be found in the corresponding subcellular compartment and hence in the corresponding daughter cell type. Support for such a mechanism has been provided by the observation that fusing the upstream regulatory region of the hook operon (which contains the f/aK gene) to a promoterless neomycin phosphotransferase reporter gene results in neomycin phosphotransferase reporter protein and translatable mRNA being found specifically in swarmer cells (Gober et al., 1991). The presence of the reporter protein in the swarmer cell is thought to reflect the localization of the expression of this gene within the predivisional cell. Thus, one aspect of the control of the asymmetry of Caulobacter cell division reduces to the question of why two chromosomes within the mother cell are differentially active. A number of mechanisms seem plausible (Figure 3b). For example, the compartment at the pre-swarmer pole might contain transcription factors that differ from those at the pre-stalked pole. Alternatively, the two chromosomes might exist in different states, resulting in differing affinities for specific transcription factors and/or in differing abilities to transcribe particular genes. Evidence that the nucleoids of stalked and swarmer cells have different sedimentation properties is consistent with this latter hypothesis (Evinger and Agabian, 1979; Swoboda et al., 1982). In either case, the question would simply be moved one step back, to the issue of what makes these differences between the two subcellular compartments. Thus, asymmetric cell division by Caulobacter involves a morphologically and molecularly polar mother cell for which it is known that aspects of this polarity causally result in the differences between the two daughter cells produced. To understand the mechanisms responsible for conferring this functionally important polarity to the mother cell, the molecules involved must be identified and charac-
Cdl 240
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4. B. subtilis
Figure based on Losick and Stragier (1991). (a) During spore formation of the bacterium B. subtilis, a vegetative cell divides asymmetrically to generate daughters of different sizes. The larger cell, known as the mother cell, engulfs the smaller cell, the forespore, which differentiates into the spore. The dashed line represents the cell wall that surrounds both daughter cells. (b) Differences in gene expression between the mother cell and the forespore result from differences in transcription factors. The oK transcription factor specifies the expression of mother cell-specific genes, and the oG transcription factor specifies the expression of foresporespecific genes. Expression of the gene that encodes oK is specified by another o factor, oE. Expression of the gene that encodes oG is specified by 19. Both oE and oF appear to be activated after the time of cell division, by the products of the .?pd/GA and spa//AA genes, respectively. (c)Cell signaling plays an important role during mother cell and spore differentiation. For example, oF activity in the forespore regulates oE activity in the mother cell; oE activity in the mother cell regulates o0 activity in the forespore; and aG activity in the forespore regulates oK activity in the mother cell.
terized. Mutations that affect the asymmetry of CaUlObatter cell division should help identify such molecules. For example, mutants defective in the gene p/eC (pleiotropit) form inactive flagella and no stalk, producing two morphologically identical daughter cells, each with a straight flagellum (Figure3c)(Sommer and Newton, 1989). Further characterization of p/eC has revealed that this gene interacts functionally with genes necessary for cell division, thereby linking the mechanisms responsible for cell division and those responsible for the generation of asymmetry (Sommer and Newton, 1991).
Bacillus subtilis When starved for nutrients, cells of the bacterium B. subtilis generate spores, which can survive harsh environmental conditions (Setlow, 1988; Kunkel, 1991). Spore formation involves asymmetric cell division (Figure 4a). A vegetative cell replicates its DNA to generate two daughter chromosomes that become separated by a septum that develops near one pole of the cell. One of the resulting compartments, called theforespore, differentiates into the mature spore. The second compartment, referred to as the mother cell, envelops the forespore and produces a protective coat that encases the spore. Mother cells are approximately four times the volume of forespore cells. Thus, during spore formation a B. subtilis cell divides to generate sister cells of two distinct types. These sister cells differ not only morphologically but also in the proteins they contain (Setlow, 1988; Zheng and Losick, 1990). In many cases these differences in proteins reflect differences in patterns of transcription, which in turn result from the effects of compartment-specific transcription factors. The genes responsible for the differences in mother cell and forespore gene expression have been analyzed in some detail (Figure 4b). For example, midway through differentiation, mother cells and forespores contain distinct transcriptional regulators: the mother cell contains a factor (oK) that recognizes promoters for mother cell-specific genes (Kunkel et al., 1989), and the forespore contains a factor ((I~) that recognizes promoters for forespore-specific genes (Sun et al., 1989). This difference in CJfactors is caused by differences in yet other o factors (reviewed by Stragier and Losick, 1990; Losick and Stragier, 1992). Thus, transcription of the gene that encodes the forespore-specific factor oG is specified by oF, which acts within the newly formed forespore (Margolis et al., 1991). Similarly, transcription of the gene that encodes the mother cell-specific factor oK is specified by another o factor, uE, which like aF acts only after the time of septum formation (Stragier et al., 1988). Interestingly, the genes that encode oF and oE are expressed in the predivisional vegetative cell, although these o factors are active only after cell division has occurred, and each is active in only one of the two sister cells. How are oF and oE activated in this cell type-specific way? The activation of oE involves proteolytic cleavage, since oE is synthesized as an inactive proprotein, the cleavage of which is controlled by the product of the spa//GA gene. The activation of oF involves two other genes, spollAA and spo//AB (Schmidt et al., 1990). These genes act in a negative regulatory cascade, such that the SpollAB protein negatively regulates the activity of oF and the SpoIlA!, protein negatively regulates the activity of the SpollAB protein. Thus, cell type-specific activity of spa//GA and spa//AA could lead to corresponding cell type-specific activity of uE and oF, respectively. Both the spa//GA and the spol/AA genes are expressed in the predivisional cell (Gholamhoseinian and Piggot, 1989). Margolis et al. (1991) have suggested that the SpollAA protein might be inactive prior to septation and activated within the forespore. If so, the question then becomes how SpollAA (and possibly SpollGA) activity changes in this way.
Review: 241
Mechanisms
of Asymmetric
Cell Division
Gene expression in both the forespore and the mother cell is regulated by signaling between these two sister cells (Figure 4c) (reviewed by Losick and Stragier, 1992). Specifically, oF activity in the forespore is necessary for the proteolytic activation of aE in the mother cell; aE activity in the mother cell is necessary for the process of engulfment, which causes oG activation in the forespore; and oG activity in the forespore is necessary for the activation of oK in the mother cell. (oK activation, like oE activation, involves proteolysis of a proprotein.) This series of signals back and forth between the forespore and the mother cell has been named “crisscross regulation” by Losick and Stragier (1992) and provides a striking example of sister cells communicating with each other during the course of their differentiation. Thus, asymmetriccell division during B. subtilissporulation results in sister cells that at the time of their formation are different in size and probably different in some key molecular features, such as the activity of oF. For this reason, the asymmetry of this cell division can be considered to be intrinsically determined. Nonetheless, the sister cells generated then interact via cell signaling to drive further differentiation by activating a cascade of transcription factors responsible for cell type-specific gene expression. One step along the pathway to mother cell differentiation involves a genomic rearrangement: an approximately 42 kt region is excised in the mother cell to generate a fusion gene that encodes the oK protein (Kunkel et al., 1990). This rearrangement contributes to, but is not necessary for, the restriction of oK activity to mother cells. Budding Yeast Each cell division of the budding yeast Saccharomyces cerevisiae produces sister cells that are morphologically and functionally different (Figure 5a) (Strathern and Herskowitz, 1979; Nasmyth, 1983). These cells differ in size (Hartwell and Unger, 1977). The larger is referred to as the mother cell, and the smaller as the daughter cell. The cell surface of the daughter cell, or bud, is entirely newly synthesized (Tkacz and Lampen, 1972). Because the daughter cell must increase in size before it can initiate cell division, the two sister cells differ in their abilities to divide (Brewer et al., 1984). They also differ in their abilities to switch from one mating type to another, a process known as mating-type interconversion. Haploid budding yeast cells are of two mating types, a and a. An a cell can change its genotypic mating type to a, and an a cell can change to a. This mating-type interconversion requires the activity of the HO gene, which encodes an endonuclease that catalyzes a genomic rearrangement at the mating-type locus and causes interconversion between the a and the a states (Strathern et al., 1982). The HO product is thus a regulator of the mating-type locus, which is itself the master regulator of cell type in yeast (Herskowitz, 1989). Mother cells, but not daughter cells, can undergo mating-type interconversion (Strathern and Herskowitz, 1979). Specifically, mother cells are competent to divide to produce cells with switched mating type in the next generation, whereas daughter cells are not (Figure 5b).
This difference between the two sister cells produced by each cell division is propagated through the yeast cell lineage. Yeast sister cells differ in their abilities to undergo mating-type interconversion because only mother cells can transcribe the HO gene (Nasmyth, 1983); if daughter cells are caused to transcribe the HOgene, these cells also can switch mating type (Jensen and Herskowitz, 1984). Transcription of the HO gene is regulated by more than a dozen regulatory proteins (reviewed by Herskowitz, 1989). Six genes, SW/l to SW16, are required for transcription of HO. Five other genes, SIN1 to SIN5, were identified because inactivation of these genes relieves the requirement for various SWI genes. Thus, these SIN genes behave as if their products negatively regulate HO transcription. Mutations in the SWI and SIN genes cause the normally asymmetric division of budding yeast to be symmetric instead (Figure 5b) (Nasmyth et al., 1987a; Sternberg et al., 1987; Kruger, 1991). Specifically, mutantsdefective in any of the SWI genes produce sister cells that are identical in the sense that neither cell can switch mating type (nonswitchable, NS). By contrast, mutantsdefective in the S/N7 or the SIN3 genes (the only SIN genes tested so far) also produce sister cells that are identical, in this case with both sisters being able to exhibit switching (switchable, S). Thus, the SWI and SIN genes are necessary for the asymmetry of budding yeast cell division with respect to competence to undergo mating-type interconversion. Gene interaction studies have indicated that the SWI and SIN genes act in a negative regulatory cascade in the order: SW15 + SIN3,4 -j SWll,2,3 + SIN1,2 + SWl4,6-, HO (Herskowitz, 1989). The products of most of the SWI and SIN genes have been characterized molecularly, leading to the following model. The SW14 and SW16 proteins, which cause HO gene expression, are transcription factors specific for genes expressed in the Gl phase of the cell cycle (such as HO) (Breeden and Nasmyth, 1987; Nasmyth and Dirick, 1991; Ogas et al., 1991). The SIN1 and SIN2 proteins are chromatin components that are thought to antagonize the activities of the SW14 and SW16 proteins (Kruger and Herskowitz, 1991; Kruger, 1991). The SWIl, SWl2, and SW13 proteins are general transcription factors that antagonize the activities of SIN1 and SIN2 (Peterson et al., 1991; unpublished data). The SW15 protein is a zinc finger transcription factor (Nagai et al., 1988) that is thought to antagonize the activities of the SIN3 and SIN4 genes, which in turn are thought to antagonize the activities of the SWII,SWl2, andSW/3genes(Herskowitz, 1989).Thismodel IS depicted in Figure 5c. How do the activities of the SWI and SIN genes lead to HOtranscription in only mother cells? One hint is provided by the observation that if SW15 protein is ectopically produced in daughter cells, daughter cells can switch mating types (Nasmyth et al., 1987b). Thus, not only is SW15 protein needed for switchtng by mother cells, but its absence normally prevents switching by daughter cells. This observation has focused attention on the question of why mother cells but not daughter cells contain functional SW15 protein. Some possibilities derive from the relative timing of SW15 protein synthesis and function.
Cell 242
a
b
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MOTHER CELL
SIN-
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DAUGHTER CELL Figure
(,I,,., ) -x+ HO
SW15 binds and antagonizes R
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SlNl,2
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SW15 must act during the Gl phase of the cell cyclethe time when HO transcription occurs (Nasmyth, 1985). Newly synthesized SW15 protein enters the nucleus just prior to Gl phase (Nasmyth et al., 1990; Moll et al., 1991). However, the SW15 gene is transcribed during late S phase, substantially later in the cell cycle (Nasmyth et al., 1987b). Thus, a cell’s functional complement of SW15 protein must be synthesized during the preceding cell cycle. These considerations suggest that SW15 protein synthesized in a dividing cell might be preferentially segregated to its mother cell rather than to its daughter cell. However, immunocytochemical studies of the localization of the SW15 protein have revealed its presence in the nuclei of both of these sister cells (Nasmyth et al., 1990). Some
5. Budding
Yeast
(a) The budding yeast S. cerevisiae divides to generate two unequally sized sister ceils. The smaller, known as the daughter cell, develops from a bud formed from the parent cell. The larger of the two sister cells is known as the mother cell. (b)Thefatesof thetwosister cellsgenerated bythe division of wild-type budding yeast can be distinguished based upon their abilities to undergo mating-type interconversion. The mother cell is competent to switch (S) (i.e., to generate progeny of the opposite mating type), whereas the daughter cell is not competent to switch (NS). In SWIt mutants, neither of the progeny cells can switch, whereas in SINmutants both of the progeny cells can switch. (c)Switching in budding yeast is driven by an endonuclease, the product of the HO gene, which makes a double-strand break at the matingtype locus. Expression of the HO gene is regulated by a cascade of proteins. One model for the action of these proteins is as follows. HO gene expression is triggered by the SW15 transcription factor, which binds to the upstream regulatory region of the HO gene and is hypothesized to displace a negative regulator (R), the function of which depends upon the SIN3 protein (Wang and Stillman, 1991). Based upon gene interaction experiments, the SIN4 protein acts at a similar step, but its mechanism of action is unknown. The SWIl, SWl2. and SW13 general transcriptional regulators are thought to be facilitated by the SWIB-DNA complex and thereby antagonize the SIN1 and SIN2 proteins, both of which are components of chromatin. With these proteins gone, the SW14 and SW16 proteins, which are transcriptional regulators that function to induce gene expression during the Gl phase of the cell cycle, can bind and activate HO transcription. In this way, SW15 activity leads to HO gene expression and to mating-type interconversion.
other mechanism must be responsible for the difference in SW15 function between mother and daughter cells. One model that has been suggested is based upon the size difference between the two sister cells (Sternberg et al., 1987; Nasmyth et al., 1990). The smaller daughter cell must increase substantially in size before it can reach the stage of the cell cycle at which HO transcription can begin (Brewer et al., 1984). Perhaps the SW15 protein segregated to both mother and daughter cells is unstable, and the longer delay in the smaller daughter cells results in the degradation of SW15 protein to an extent that HO transcription cannot take place (an idea challenged by Nasmyth et al. (1990)). Alternative models are also possible, and the basis of the differing levels of SW15 activity between mother and daughter cells remains a major issue. Thus, the difference between the abilities of budding
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Mechanisms
a
of Asymmetric
Cell Division
b
an
0 d
\
nn
FISSION
YEAST Figure
C
S
NS
d
yeast sister cells to undergo mating-type interconversion is controlled by a negative regulatory cascade of proteins that control the expression of an endonuclease that specifies cell type. Current data indicate that the SW15 protein acts at the earliest known point in this cascade, and it seems likely that differences in SW15 activity between mother and daughter cells are conferred intrinsically, at the time of cell division. Nonetheless, later stages of the differentiation of the a and a cell types involve cell interactions, as each mating type produces a diffusible sexual pheromone that influences the differentiation of the other (reviewed by Herskowitz, 1989). This two-step process of making sister cells intrinsically different and then reinforcing those differences by cell interactions between these cells is similar to that used for the control of asymmetric cell division by B. subtilis, as described above. Fission Yeast Each cell division of the fission yeast Schizosaccharomyces pombe produces sister cells that appear to be morphologically identical. Nonetheless, these cells have dif-
6. Fission
Yeast
(a) The fission yeast S. pombe divides to generate one daughter cell that is competent to undergo mating-type interconversion (S) and one daughter cell that is not competent (NS) (Miyata and Miyata, 1981). (b) A fission yeast cell that switches generates one daughter of a different mating type and one daughter of the same mating type as its parent cell (Miyata and Miyata, 1981). For example, this diagram shows a cell of mating type M dividing to produce one S and one NS cell. The S cell, also of mating type M, produces one cell of mating type M and one cell of switched mating type P. The NS cell produces two cells of mating type M. By contrast, a budding yeast cell that switches generates two daughters that differ in mating type from their parent. For example, a cell of mating type a divides to produce one S and one NS cell. The S cell, also of mating type a, produces two cells of mating type a. The NS cell produces two cells of mating type a. (c) Model for how DNA strand modification can confer differences between sister cells (modified from Klar, 1987). A parent cell with one DNA strand modified (solid ellipse) at the mating-type locus (arrows) will produce one daughter (on the left) with this modified DNA strand and one daughter (on the right) in which the newly synthesized corresponding DNA strand is unmodified. Only the daughter cell with the modified DNA strand is competent to undergo mating-type interconversion. The solid and open arrows represent the two complementary DNA strands. The thick and thin arrows represent old and new DNA strands, respectively. (d) An inverted tandem duplication of the mating-type locus allows both DNA strands to be modified and hence both daughter cells to undergo mating-type interconversion (modified from Klar, 1990).
ferent fates, since they differ in their abilities to undergo the process of mating-type interconversion in their next cell division(Miyataand Miyata, 1981). Like budding yeast, fission yeast has two mating types, which in this case are named P (plus) and M (minus). P cells can generate M cells, and M cells can generate P cells This mating-type interconversion involves a genomic rearrangement that is in some respects mechanistically similar to that involved in mating-type interconversion in budding yeast (Egel et al., 1984). Of a pair of fission yeast sister cells, one can switch mating type (the S cell in Figure 8a) and the other cannot (the NS cell) (Miyata and Miyata, 1981). This phenomenon is similar to that seen in budding yeast, in which the mother cell, but not the daughter cell, can switch. There are two striking differences between fission yeast and budding yeast in mating-type interconversion. First, in fission yeast a cell that switches (S) generates cells of the two different mating types (Figure 8b). (A cell that does not switch [NS] generates two cells of the same mating type as the parent cell.) For example, an M cell that switches generates one M and one P cell. By contrast, in budding
Cell 244
yeast, a cell that switches generates two cells of the opposite mating type. Thus, an a cell that switches generates two a cells. These differences probably arise from differences in the time during the cell cycle when the genomic rearrangement occurs: if rearrangement precedes replication of the mating-type locus, both daughters should switch, as in budding yeast, whereas if rearrangement follows replication, only one daughter should switch, as in fission yeast. The second and more significant difference between fission and budding yeasts concerns the basis for the asymmetry of their divisions (Klar, 1987). As discussed above, budding yeast sister cells differ in their abilities to produce the endonuclease that initiates genomic rearrangement (Nasmyth, 1983; Jensen and Herskowitz, 1984). By contrast, fission yeast sister cells appear to differ in their substrates for genomic rearrangement, namely, in the chromosomes that carry that mating-type locus. This chromosomal difference was discovered by Egel (1984), who found in studies of diploid fission yeast cells that the two homologous chromosomes containing the matingtype locus could switch independently. This observation suggests that the difference in switching competence between sister cells is not conferred by cytoplasmic differences (e.g., in an enzyme such as an endonuclease), but rather by differences in the state of the mating-type locus itself. Subsequent studies have supported the hypothesis that the substrate for genomic rearrangement is different in the two sister cells produced by the division of fission yeast. Klar(1987) proposed that a modification of one DNAstrand (e.g., by methylation) occurs at the mating-type locus (Figure 8~). Because DNA replication is semiconservative, this marked strand will generate a marked daughter chromatid that differs from its sister chromatid, in which the corresponding DNA strand is newly synthesized and hence unmodified. The marked and unmarked chromatids will be segregated to the two daughter cells, causing those cells to be molecularly distinct and thereby allowing only one, for example that with the marked chromatid, to produce progeny that switch. The newly synthesized and unmodified strand in the other cell could be modified subsequently in the cell cycle, thereby allowing this cell, like its parent cell, to generate one daughter that produces progeny that switch and one that does not. This model predicts that if two copies of the mating-type locus are present in opposite orientations, a site for modification should exist on both strands and thus allow both daughters to produce progeny that can switch (Figure 6d). Indeed, Klar (1990) observed that an inverted tandem duplication at the mating-type locus causes sister cells that would otherwise be different to instead be the same in this way. Although it is appealing to think that the DNA modification that occurs at the mating-type locus is covalent, it remains possible that this modification is noncovalent, involving for example the binding of a protein specific for one DNA strand and available at only certain times of the cell cycle. The normally asymmetric cell division pattern of fission yeast can also be caused to be symmetric hy mutation. A
variety of swi genes (which bear no known relationship to the SW/genes in budding yeast) have been identified that are necessary for switching (Egel et al., 1984). Mutants in these genes produce sister cells, neither of which can undergo mating-type interconversion. In summary, asymmetric cell division by fission yeast appears to be a consequence of the asymmetry that results from semiconservative DNA replication. Fission yeast, like budding yeast, produces mating pheromones and other molecules that function in cell interactions during mating (reviewed by Marsh et al., 1991). Thus, it seems likely that fission yeast, like budding yeast (see above), uses signaling between cells of opposite mating type to lead to further differentiation of two sister cells that are intrinsically different. Nematode Of the 949 nongonadal cell divisions that occur during the development of the nematode Caenorhabditis elegans, 807 are asymmetric; that is, these divisions generate daughter cells that either differentiate in different ways or that divide to generate different sets of descendant cells (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979; Sulston et al., 1983, 1988). The C. elegans asymmetric cell division that has been analyzed in the most detail is the first cleavage of the egg. The asymmetry of this first cell division seems likely to be intrinsically determined (reviewed by Strome, 1989). We summarize below what is known about this cell division to exemplify the types of experimental approaches that have been taken. We choose this example from among many that have been studied using the methodologies of experimental embryology (e.g., Davidson, 1986) because it is being subjected to a genetic analysis that promises to reveal the molecules and mechanisms responsible for an intrinsically determined asymmetric cell division (Kemphues et al., 1988). The C. elegans egg divides to form two differently sized daughters, a larger anterior cell called AB and a smaller posterior cell called PI (Figure 7a). Thus, these sister cells are different at the time they are generated. AB and P, are clearly distinct in their fates. First, they undergo different patterns of cell division: AB displays a series of synchronous cleavages, each of which produces sister cells that are equal in size, whereas PI is a stem cell with descendants that are asynchronous in their times of division and unequal in their sizes. Second, AB and PI generate different cell and tissue types: AB produces primarily ectoderm, whereas PI produces mesoderm, endoderm, and the gonad, as well as some ectoderm. A number of observations indicate that AB and PI have distinct developmental potentials. First, studies of isolated AB and PI cells indicate that differences between the fates of these cells do not depend upon interactions either between them or among their descendants. Laufer et al. (1980) and Edgar and McGhee (1986) squashed eggs to lyse either AB or P,, and Priess and Thomson (1987) surgically removed either AB or PI using a microneedle. In the resulting partial embryos, an isolated AB cell generates equally sized daughters that divide synchronously to produce many descendants that become ectodermal cells, as
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(b) Model: cytoplasmic
Cell Division
determinants
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(d) g/p-7 mutants
f Figure
7. Nematode
Embryonic
Cell Divisions
(a) The C. elegans egg divides asymmetrically to generate a larger anterior daughter AB and a smaller posterior daughter PI. AB and P, differ in many ways. AB then divides asymmetrically to form an anterior daughter ABa and a posterior daughter ABp, while P, divides asymmetrically to form an anterior daughter EMS and a posterior daughter Pz. ABa, ABp, EMS, and PI all differ in their fates. (b) One model for the role of cytoplasmic determinants in the first egg division. Determinants responsible for specifying the P, fate are concentrated within the posterior cytoplasm of the egg and segregated preferentially to the posterior daughter cell. (c) Mutations in the par genes cause the first zygotic cell division to be symmetric instead of asymmetric, generating two nearly identical sister cells that fail to produce certain PI descendants and can produce certain AB descendants in excess, suggesting that the two daughter cells are both AB-like in character. (d) Mutations in the g/p-l gene appear to cause the two daughters of AB to both express the ABp fate. (e) Two models for the role of the Glp-1 protein, which has been postulated to be a cell surface receptor, in the cell interactions responsible for making the AB cell divide asymmetrically. In both models, a signal from EMS causes the anterior AB daughter to express the ABa fate instead of theABpfate. In thefirst model,shownabove, theGlp-I protein isthereceptorfortheEMSsigna1. In thesecond model, shown below, aGlp-l-mediated signal from the posterior daughter acts in conjunction with a signal from EMS to elicit the ABa fate. In either case an absence of EMS signal or of Glp-1 receptor would cause the anterior daughter to express the ABp fate.
in the intact embryo. An isolated PI cell generates unequally sized daughters that divide asynchronously to produce descendants that twitch and contain intestinespecific markers, again as in the intact embryo. Similarly, after cytokinesis has been blocked using cytochalasin, a PI cell expresses muscle-specific and intestine-specific
markers, while A6 does not (Laufer et al., 1980; Cowan and McIntosh, 1985; Edgar and McGhee, 1988). Together, these studies establish that a number of the differences between the descendants of AB and those of P, are determined by the two-cell stage and do not depend upon interactions between AB and PI.
Cell 246
The difference in developmental potential between AB and PI appears at least in part to be conferred by a cytoplasmic factor (Figure 7b), since the cytoplasm of PI seems to be able to induce expression by AB of intestine-specific markers: embryos in which the P, nucleus was extruded through a hole made by laser microsurgery and in which the cytoplasms of AB and PI were fused displayed the autofluorescence characteristic of differentiated intestinal cells (Schierenberg, 1965). In addition, the potential of the egg to divide to produce two unequally sized daughters might be conferred by activity localized to its posterior cytoplasm, since extrusion of its posterior but not of its anterior cytoplasm has been reported to cause the egg to produce equally sized daughters (Schierenberg, 1965). Although the factor(s) responsible for conferring these differences between AB and P, has not been identified, cytoplasmic elements are known to be asymmetrically segregated during cleavage of the C. elegans egg, establishing that an appropriate mechanism exists for the segregation of developmental determinants. Specifically, P granules, which are found in germ cells at all developmental stages, are localized to the posterior pole of the egg and upon division segregate to P, but not to AB (Strome and Wood, 1962). Together these observations suggest that differences between AB and PI might well be conferred by the differential segregation of factors asymmetrically localized within the egg. What mechanisms are responsible for the localization of cytoplasmic factors within the nematode egg? During its first cell cycle, the fertilized egg undergoes a major cytoplasmic reorganization (Albertson, 1964) similar to that seen in many other embryos and believed to be responsible for cytoplasmic localization (Davidson, 1966). The treatment of C. elegans embryos with microfilament inhibitors (but not with microtubule inhibitors) disrupts this cytoplasmic reorganization, as well as P granule segregation and other indications of egg cell polarity, suggesting that actin microfilaments play a role in establishing this polarity (Strome and Wood, 1963; Hill and Strome, 1990). To identify other molecules that function in controlling cytoplasmic localization within the egg, Kemphues and coworkers (1966) have taken a genetic approach. Eight maternal effect lethal mutants defining four par (partitioning-defective) genes fail in P granule localization and other aspects of cytoplasmic reorganization (Kirby et al., 1990). These mutants in general produce AB and PI cells of similar sizes, which divide at the same time and continue dividing synchronously. Intestinal and germline cells, which are normally derived from PI, are missing, whereas muscle cells normally derived from AB are produced in excess in at least some par mutants. These and other observations have led to the suggestion that in par mutants the egg divides to form two nearly identical sister cells that both express an AB-like or a hybrid ABlP, fate (Figure 7~). A reasonable hypothesis is that the par genes function in cytoplasmic localization within the egg and are necessary for the determination of the PI cell fate. Further analysis of these and similar genes promises to identify the molecules responsible for these processes.
Asymmetric Ceil Divisions by Cell Signaling
Determined
How can two sister cells that are initially identical in their developmental potentials become different? First, one or both of the sister cells could respond to signals from other cells (see Figure 2~). Intercellular signaling is common in the development of many organisms (Gurdon, 1967; Pawson, 1991; Gurdon, 1992, this issue; Jesse11 and Melton, 1992, this issue), and signaling that influences 1 of 2 equipotent sister cells could act via mechanisms and molecules like those involved in other aspects of intercellular communication. Alternatively, stochastic events could cause two sister cells to differ in a small way that might be reinforced by a feedback mechanism involving signaling between the two sister cells (see Figure 2d). Many examples of sets of cells that are equivalent in developmental potential but that express different fates as a consequence of cell interactions are known. Such sets of cells are called “equivalence groups” and were first identified and have been most extensively characterized in C. elegans(Kimble et al., 1979; Sulston and White, 1960; Sulston, 1966; reviewed by Greenwald and Rubin, 1992, this issue). We discuss below examples of equivalence groups that consist of sister cells. Nematode The two daughters of the C. elegans AB blastomere constitute an equivalence group. The anterior daughter ABa and the posterior daughter ABp (Figure 7a) differ drastically in their fates, generating different numbers and types of cells and undergoing distinct patterns of cell division (Sulston et al., 1963). For example, ABa generates muscles of the pharynx, whereas ABp does not. Priess and Thomson (1967) used a micromanipulator to exchange the positions of ABa and ABp and found that the fates of these cells were then reversed. Normal and fertile animalsdeveloped, establishing the completeness of the transformation of the fates of these cells. This experiment revealed that each of these two cells when generated has the potential to express either the normal ABa or the normal ABp fate; which fate is expressed depends on cell position, presumably as a consequence of cell signaling. Priess and Thomson (1967) also found that after puncturing the eggshell and squeezing the EMS cell (Figure 7a) through the resulting hole, ABaderived pharyngeal muscles did not form. This observation suggests that EMS or its descendants induce ABa or its descendants to generate pharyngeal muscle, and that the different fates of the sister cells ABa and ABp are at least in part a consequence of interactions with another cell or cells. One molecule that functions in the determination of the ABa fate and is likely to act in cell signaling has been identified by genetic studies. Priess et al. (1967) isolated four mutants that fail to generate ABaderived pharyngeal muscle cells and that have other characteristics like those seen in embryos that lack the EMS cell (Figure 7d). All four mutants are abnormal in the same gene, g/p-l (germline proliferation defective), which encodes a member of a con-
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Figure based on Kuwada and Goodman (1985). (a) The asymmetric division of the ganglion mother cell GMC-1, a daughter of neuroblast NB l-1, generates sister cells that differentiate into two distinct neuron types, the anterior corner cell aCC and the posterior corner cell pCC. (b) The asymmetric division of the midline precursor cell MP3 generates sister cells that differentiate into two distinct neuron types, known as the H cell (because of its H-like shape) and the H cell sib.
served protein family that includes the products of the C. elegans gene lin-12 and of the Drosophila gene Notch (Yochem and Greenwald, 1989; Austin and Kimble, 1989; reviewed by Greenwald and Rubin, 1992). These proteins seem likely to act as cell surface receptors that function in intercellular signaling via direct cell contact (Kidd et al., 1989; Fehon et al., 1990). One possibility is that ABa is different from its sister ABp as a result of a signal from EMS that is received by Glp-1 protein located on the surface of ABa (Figure 7e). Alternatively, a signal from EMS and a parallel signal (possibly from ABp) received by Glp-1 protein located on the surface of ABa might be required together for the expression of the ABa fate. Grasshopper Two examples of sister cell equivalence groups were discovered during an analysis of the embryonic development of the grasshopper nervous system (Kuwada and Goodman, 1985). First, the two daughters of the ganglion mother cell GMC-1 differentiate into the morphologically clistinct anterior and posterior corner cells, known as the aCC and pCC neurons, respectively (Figure 8a). When one of these two daughter cells, chosen at random, was killed using an intracellular microelectrode, the remaining cell acquired characteristics of the pCC neuron in 8 of 9 cases. This experiment indicated that both cells can express the pCC fate and suggested that at the time they are formed, the two sister cells are equivalent in their developmental potentials. Similarly, the midline precursor cell MP3 divides asymmetrically to generate two morphologically distinct neurons, the H cell (which is shaped like the letter H) and the l-i cell sib (Figure 8b). When one of these two cells was killed using a microelectrode, the surviving cell generally expressed characteristics of the H cell sib. Thus, both of these cells appear to have the potential to express the H cell sib fate and thus are likely to constitute a sister cell equivalence group. Whether the more anterior or the more posterior of the two sisters becomes the H cell sib varies randomly, which supports the hypothesis that the cells are equivalent in their developmental potentials. The sister cells in each of these two cases must interact, since when both cells are present, only one expresses the
230 V
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9. Anabaena
(a) Vegetative (V) cells of at least one strain of Anabaena can divide asymmetrically to produce a larger daughter that differentiates into a vegetative cell and a smaller daughter that differentiates into a nitrogen-fixing heterocyst (H). (b) The asymmetry of Anabaena cell division can be influenced by intercellular signaling. A signal (dashed line) from a nearby heterocyst can cause a presumptive heterocyst to differentiate into a vegetative cell.
potential that it would express if the other cell were absent. In principle, this interaction could be either active or passive. For example, a cell expressing one fate could signal its sister to prevent it from expressing that fate. Alternatively, there could be a competition between the sister cells for proximity to (or distance from) the source of a signal that determines which of the two fates the cells will express. This latter mechanism is common in non-sister cell equivalence groups studied in C. elegans. For example, the ectoblast cells P5/8L and P5/8R constitute an equivalence group (reviewed by Horvitz and Sternberg, 1991). These two cells are generated on the left and right sides of the animal, respectively, and then centralize, so that one becomes located anterior to the other. Their subsequent fates depend on their anterior-posterior positions, as a consequence of their having assumed different positions with respect to a signal from a nearby cell, the anchor cell of the gonad. Cyanobacteria Cyanobacteridof the genus Anabaena grow as filaments of identical vegetative cells that divide about once a day (Wilcox et al., 1975). Heterocystous cyanobacteria respond to an environment with limiting nitrogen by generating terminallydifferentiatedcellscalled heterocysts, which function in nitrogen fixation and differ from vegetative cells in many morphological and biochemical respects. A new heterocyst will develop along a growing filament at a position approximately midway between adjacent preexisting heterocysts, maintaining a pattern in which heterocysts occur at approximately equal intervals along a filament. Since vegetative cells neighbor each heterocyst, asymmetric cell divisions cause certain vegetative cells to divide to generate one vegetative cell and one heterocyst (Figure 9a). What causes a heterocyst to become different from its vegetative cell sister? Vegetative cells of one strain of Anabaena divide to form sister cells of different sizes (Wilcox et al., 1975), and heterocysts develop from only the smaller of these cells. Since this difference in cell size is defined
Cdl 240
by the location of the septum in the mother cell, it would seem that this asymmetric cell division is intrinsically determined. However, not all of the smaller daughters differentiate into heterocysts. Specifically, only those small cells sufficiently distant from nearby heterocysts differentiate into heterocysts. Furthermore, if a developing heterocyst is punctured with a microneedle, a new heterocyst will form nearby (Wilcoxet al., 1973). These observations suggest that heterocyst formation is inhibited by signals from other heterocysts (Figure 9b). Since heterocysts occur periodically along a filament (about every five or six cells in the strain studied by Wilcox), this inhibitory signal can act at a distance. This role of cell signaling in asymmetric cell division contrasts with that seen in nematodes and grasshoppers, as described earlier in this section: in nematodes and grasshoppers cell signaling causes sister cells that are initially identical to become different, whereas in Anabaena cell signaling prevents sister cells that are initiallydifferent (at least in size) from becoming different cell types. Buikema and Haselkorn (1991a) have identified a mutant that fails to differentiate heterocysts from among a collection of mutants defective in nitrogen fixation. This mutant is defective in the hetR gene, which encodes a novel protein with no recognized sequence motifs (Buikema and Haselkorn, 1991 b). Overexpression of the herfi gene causes an increased frequency of heterocyst formation, even on media containing nitrogen. These observations indicate not only that hetR function is necessary for heterocyst formation, but also that the addition of hetR function is sufficient to cause cells that would not normally become heterocysts to do so. In other words, hetf? controls heterocyst formation. It seems likely that hetR regulates two DNA rearrangements that occur during heterocyst formation (Golden et al., 1985,1988), although thisregulation could be quite indirect. Since heterocysts can form in the absence of at least one of these rearrangements (Golden et al., 1988), these rearrangements could be consequences rather than causes of heterocyst differentiation. volvox Development of the spherical multicellular green alga VolVOX carteri forma nagariensis involves asymmetric cell divisions that produce cells of groksly differsnt sizes and strikingly different fates (reviev+ by Kirk et al., 1991). Whether the asym intrinsically or by cell i less, it is clear that t which cells ciivlde division, and hence the asymmetrically, is contr sexual hormones. Development of the asexual forni of Volvox carteri forma nagariensis begins with five symmetric cell divisions, which generate 32 cells of nearly equivalent size. The sixth cell division of the 18 anterior cells is asymmetric, generating a small anterior daughter and a large posterior daughter (Figure lOa). The 18 large cells divide asymmetrically twice more, in each case forming a small and a large daughter. The 16 terminal large daughters differentiate into the 16 asexual reproductive or gonidial cells of the
adult. These large cells are known as gonidial initials. All other cells produce only somatic cells, and are known as somatic initials. The somatic initials generate the total of about 2000 somatic cells found in the adult. The symmetry of specific cell divisions can be influenced by cell signaling (Figure lob). Although male and female strains are morphologically indistinguishable in their asexual phases, these strains are visibly dimorphic in their sexual phases. A pheromone produced by mature sexual males causes cells in the sixth round of cell division in developing female embryos to divide symmetrically instead of asymmetrically. Asymmetric divisions occur in the seventh round instead. Furthermore, more cells divide asymmetrically in such pheromone-treated females than in individuals not exposed to pheromone. The smaller cells are somatic initials, and the larger cells differentiate into eggs. Similarly, in sexually induced male embryos, asymmetric cell division is suppressed in the sixth, seventh, and possibly eighth rounds of cell division. Only the last round of cell division is asymmetric, generating equal numbers of somatic cells and precursor cells that divide symmetrically to produce packets of 64 or 128 sperm cells. During both normal asexual and sexual development as well as under a variety of abnormal conditions (e.g., after heat shock, subsequent to surgical manipulation, and in mutants), cell size and cell fate are strongly correlated, with larger cells invariably becoming reproductive cells. This observation has led to the hypothesis that it is the difference in the sizes of sister cells that is responsible for the difference in their fates (Pall, 1975; Kirk et al., 1991). Mutantsof Volvox have been isolated in which asymmetric cell divisions do not occur (Figure 10~). Because these mutants fail to generate gonidia, they are known as gonidia-less and define the gene g/s. Kirk et al. (1991) propose that the product of the gls gene functions in the placement of the Volvox mitotic apparatus and that when this gene is defective, the mitotic apparatus by default becomes located at the middle of the cell. Genes Required Cell Divisions
for the Asymmetry
of Specific
Genetics offers a powerful approach toward the elucidation of mechanisms that make sister cells different. Mutants in which sister cells that normally express different fates instead express the same fates can define genes that function in making sister cellsdifferentfrom each other. An analysis of such genes and their products should reveal the basis of their actions. For example, as discussed above, C. elegans mutants that cause the cell ABa to have characteristics of its sister cell ABp are altered in the Glp-1 protein, which is probably a cell surface receptor. This observation indicates that differences between ABa and ABp are most likely caused by cell signaling. A number of genes have been identified both in C. elegans and in Drosophila that are required for the asymmetry of specific cell divisions. Whether the sister cells generated by those divisions are different when they are formed or become different afterward is not yet known. The study of these genes should indicate not only whether or not
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these asymmetric cell divisions are intrinsically determined, but also should lead to an understanding of the complete genetic and biochemical pathways that are responsible for making these sister cells different. Nematode Studies of mutants abnormal in the development of the vulva of the C. elegans hermaphrodite have identified four genes necessary for the asymmetry of specific cell divisions (Ferguson et al., 1987). Mutations in each of these genes perturb the asymmetry of 1 of 2 sequential cell divisions involved in vulva1 development (Figure lla). The vulva is generated by three ventral ectodermal blast cells known as P5, P6, and P7. Each of these three P cells divides asymmetrically to form an anterior neuroblast (Pn.a cell, shown as NB in Figure lla) and a posterior ectoblast (Pn.p cell, shown as ECT). Two of the posterior ectoblasts (P5.p and P7.p) then divide asymmetrically to form two different classes of ectoblast cells (LL, TN), which are distinguished by both their subsequent division patterns and the numbers and types of descendant cells they generate. In /in-26 (lineage abnormal) mutants, the divisions of the P cells are symmetric instead of asymmetric: the two sister cells generated are both neuroblasts, apparently because each posterior daughter expresses the fate normally expressed by its sister (Figure 11 b) (Ferguson et al., 1987). Mutants defective in the gene In-1 7 have a similar effect
0 ++
10. Volvox
Figure based on Kirk et al. (1991). (a) During asexual development of individuals of either genotypic sex, asymmetric cell division commences with the sixth round of cell division and continues through the eighth round. The large daughter cells generated by the eighth round of cell division differentiate into the asexual reproductive cells of the adult. (b) The symmetry of cell division is influenced by a sexual pheromone (dashed fine), which is produced by sexually mature males and by heat-shocked asexual individuals. This pheromone delays the onset of asymmetric cell division to the seventh round in female zygotes and to the eighth or ninth round in male zygotes. The large daughter cells generated by the seventh round in female zygotes become eggs, and those generated by the eighth or ninth rounds in male zygotes become precursor cells that each generate 64 or 128 sperm. (c) In the g/s mutant, all cell divisions are symmetric in both asexually and sexually developing zygotes.
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on the subsequent asymmetric cell division: two of the ECT cells (P5.p and P7.p) divide symmetrically instead of asymmetrically, as both daughters express the LL fate (Figure 11 c). Mutants defective in the /in-l 7or /in-78 genes variably disrupt the asymmetry of one of these ECT cell divisions (by P7.p). In some individuals this division is symmetric, generating two daughters that express the same abnormal (ABN) fate, which is different from both of the fates normally expressed by the two daughters of this division (Figure 11 c). In other /in-l 7 or /in-78 individuals this ECT cell division is reversed in its polarity, so that the anterior daughter expresses the fate normally expressed by the posterior daughter, and vice versa. Since the h-26, h-7 7, h-1 7, and lin-78 mutations characterized all reduce or eliminate gene activity (Ferguson and Horvitz, 1985), the functions of these four genes are normally needed to make certain sister cells different from each other. The effects of h-77 have been characterized further (Sternberg and Horvitz, 1988). Mutations in /in-77 not only affect the vulva1 cell lineages but also cause at least five other types of ectodermal and gonadal blast cells that normally divide asymmetrically to divide symmetrically instead. In addition, /in-77 mutations cause sister cells to be equal in size as well as equivalent in developmental fate. These observations suggest that the M-7 7 product acts in mother cells as a component of a mechanism that establishes the asymmetry of specific asymmetric cell divisions. The asymmetric cell divisions affected by /in-l 7, /in-77,
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(a) Two sequential asymmetric cell divisions occur during the postembryonic development of the C. elegans vulva (Sulston and Horvitz, 1977; Sternberg and Hot-&z, 1986). First, each of three P ectodermal blast cells P5-P7 divides to generate an anterior neuroblast (Pna, called NB in this figure) and a posterior ectoblast (Pn.p, called ECT). Two of these ectoblasts, P5.p and P7.p, then divide to generate two different types of ectoblasts, the anterior of which is known as LL and the posterior of which is known as TN. (b) In the fin-26 mutant, the sister cells generated by the P cells both become neuroblasts (Ferguson et al., 1987). (c) In /in-l7 mutants, the sister cells generated by P5.p and P7.p both become LL ceUs (Ferguson et al., 1987). In M-77 and /in-78 mutants, the sister cells generated by P7.p often have identical fates that are different from both of the normal fates of the daughters of this division. (d) During embryonic development of the C. elegans nervous system, an asymmetric cell division generates the PHB sensory neuron and the HSN motor neuron (Sulston et al., 1983). By contrast, a ham-f mutant seems to generate two identical sister cells that have characteristics of both the PHB and HSN neuron types (Desai et al., ISEIS).
and /in-18 seem likely to be intrinsically determined, since the fates of the daughter cells produced have not been altered by the killing of other cells. For example, the two sister cells (LL and TN) generated by the divisions of the P5.p and P7.p cells do not seem to constitute an equivalence group, since the killing of one does not cause the other to express its fate (Sternberg and Horvitz, 1988). However, the failure of such experiments to reveal cryptic developmental potentials necessarily constitutes negative evidence, and it remains conceivable that cell interactions function in specifying these cell fates. The variable abnormalities seen in /in-17 and /in-18 mutants could be explained if mutations in these genes perturb cytoplasmic localization within the mother cell so that either the polarity of the distribution of specific developmental determinants is reversed or such determinants become mixed causing
both daughter cells to express the same, but abnormal, fate. The gene lin-ll encodes a putative transcription factor, which contains a homeodomain and two copies of a cysteine-rich motif called LIM (Freyd et al., 1990). It seems likely that the Lin-1 1 protein acts to cause differential gene expression between the sister cells generated by the divisions of the P5.p and P7.p cells, and that these differences in gene expression result in the different fates of these sister cells. What might cause /in-l1 to be active in only one of the two sister cells LL and TN? One possibility emerges from the observation that the LIM domain of the Lin-11 protein binds two atoms of zinc and four atoms each of iron and free sulfur in a redox-sensitive iron-sulfur cluster (Li et al., 1991). Thus, Lin-I 1 activity might be regulated by changes in redox potential that occur during the course of the vulva1 cell lineages. In principle, h-11 might act in the mother cell to produce RNAs or proteins that are differentially distributed to the two daughter cells, might be expressed in the mother cell with either its own RNA or protein products being asymmetrically distributed to one of the two daughter cells, or might be expressed in only one of the two daughter cells. Since /in-l 1 affects the same cell division in vulva1 development as does/in-l 7, the/in-l 7 (and also the /in-18) gene might act in the mother cell with In-17 or act in one of the daughter cells in response to /in- 17. The C. elegans gene ham-l (HSN abnormal migration) also affects asymmetric cell division (Desai et al., 1988). ham-l was discovered in a screen for mutants abnormal in the two serotonergic HSN motor neurons, which are required for egg laying. In ham-l animals, the presumptive HSN neurons do not undergo their normal migrations apparently because they fail to acquire a proper HSN identity. ham-l mutants often have two extra HSN neurons, Interestingly, ham-l mutants also often lack the sisters of the HSN neurons, the PHB sensory neurons, which suggests that the PHB neurons are transformed to express the fate of their sister cells. However, in ham-l animals the HSN neurons are abnormal, presumably because they are partially transformed into PHB neurons. Thus, the two daughters of each PHB-HSN mother cell seem to express characteristics of both the PHB neuron and the HSN neuron (Figure lld). One plausible interpretation of the Ham-l phenotype is that the asymmetric division of the PHB-HSN mother cell is intrinsically determined and the distribution of developmental determinants from the mother cell to its daughters is perturbed in ham-l animals. If so, ham-l functions in the localization or segregation of developmental determinants. Alternatively, if the PHB-HSN division is regulated by cell interactions, ham-l could function in a cell signaling process responsible for determining the fates of sister cells. Two other C. elegans genes that function in asymmetric cell division warrant mention. Mutations in these genes change an asymmetric cell division of one type into an asymmetric cell division of another type. The /in-44 gene, identified on the basis of its effects on the development of the male tail, specifies the polarity of certain asymmetric cell divisions, i.e., in /in-44 animals sister cells express
Review: 251
Mechanisms
of Asymmetric
(a) wild
Cell Division
type
(b) numb
mutants
0
-+ 60 S
(c)
S
oversensitive
mutants
0 d GN
Figure
12. Drosophila
Figure based on Uemura et al. (1989) and Jan and Jan (1990). (a) In the embryonic development of the peripheral nervous system of Drosophila, the precursor cell that generates an es organ appears to divide asymmetrically to form one structural cell precursor(S) and one precursor that generates a glial cell and a neuron (GN). (b) In the numb mutant, the es precursor cell generates two structural cell precursors. (c) In the oversensitive mutant, the es precursor cell generates two glial-neural precursors.
each other’s normal fates (Horvitz, 1990; Herman, 1991). Thus, if an anterior daughter normally is of type A and the posterior daughter of type B, in a /in-44 mutant the anterior daughter can be of type B and the posterior daughter of type A. In-44 could act either before cell division to define the polarity of the mother cell or after cell division to define the fates of the daughter cells. The unc86(uncoordinated) gene, which was discovered because it affects a number of behaviors, causes one of the two sister cells generated by the asymmetric division of certain neuroblasts to become different from its mother cell (Horvitz and Sulston, 1980; Chalfie et al., 1981). Thus, if a mother cell of type C normally divides to make daughters of types A and B, in an uric-86 mutant that cell can divide to make daughters of types A and C. The Uric-86 protein is a member of the POU family of transcription factors and is expressed specifically within the daughter cell it affects, suggesting that it controls gene expression in that cell to make it different from its mother (Finney et al., 1988; Finney and Ruvkun, 1990).
Fruit Fly Studies of the development of the Drosophila nervous system have led to the identification of two genes that appear to function in asymmetric cell division. Mutants defective in the numb gene lack most of the neurons of the embryonic peripheral nervous system and have more than the normal number of support cells usually associated with these neurons (Uemura et al., 1989). For example, a simple external sensory or “es” organ, which is thought to be chemosensory or mechanosensory in function, normally consists of one neuron, one glial cell, and two support cells. These four cells are probably generated by two rounds of cell division by a single precursor cell. In numb animals, four cells are present in each es organ, but all four are support cells. Since the two support cells seem to be derived from the sister of the cell that generates the glial cell and neuron, numb mutations probably transform the glial-neuroblast to express the fate of its sister cell, the support cell precursor (Figure 12). It is not known whether the asymmetric division of the es organ precursor cell is intrinsically determined or whether the two daughter cells it produces become different as a consequence of cell interactions. Interestingly, mutations in the oversensitive gene have effects that are opposite to those of mutations in thenumb gene (Jan and Jan, 1990). Specifically, loss of function of the oversensitive gene causes the support cell precursor to express the fate of the glial-neuroblast precursor (Figure 12). Thus, each of these sister cell fates requires the function of a specific gene for its expression, so that neither fate can be regarded as a simple default state. Presumably, the numb and oversensitive genes act in a negative regulatory pathway that specifies the fates of these sister cells, as do the SW/ and SIN genes in budding yeast. The order of action of these Drosophila genes within such a pathway should be revealed by further genetic analyses. Conclusions Although the fundamental basis of asymmetric cell division is not yet understood at a mechanistic level for any of the examples described above, enough is known to establish that a variety of distinct mechanisms are used to make sister cells different from each other. In some cases sister cells are different from the time they are generated, whereas in other cases initially identical sister cells become different as a consequence of cell interactions. The intrinsic differences between sister cells can be determined either by differences in their cytoplasmic factors (B. subtilis, budding yeast, and the nematode AB and PI cells) or by differences in their chromosomes (fission yeast, possibly Caulobacter and Anabaena). It should be noted that these mechanisms need not be mutually exclusive, and it remains possible that both cytoplasmic and chromosomal differences function in certain asymmetric cell divisions. Cell signaling seems to be able to affect the asymmetry of cell division either via direct cell-cell contact (nematode ABa and ABp cells) or via a diffusible molecule (Volvox). Cell signaling can even act to cause intrinsically asymmetric cell divisions to be symmetric instead (Anabaena, pos-
Cdl 252
sibly Volvox). In addition, cell signaling can play a role in inducing further differentiation of sister ceils that are intrinsically determined to be different (6. subtilis, budding yeast, possibly fission yeast). The examples we have discussed reveal that many classes of molecules function to make sister cells different from each other. Transcription factors, and other DNAbinding proteins such as components of chromatin, act in 1 of 2 sister cells to regulate differentially the expression of batteries of genes responsible for particular differentiated characteristics (B. subtilis, budding yeast, nematode). Such transcription factors are themselves regulated by other cell type-specific transcription factors (B. subtilis). Nucleases catalyze genomic rearrangements and proteases activate proproteins in processes necessary for the differentiation of 1 of 2 sister cell types (B. subtilis, budding yeast, fission yeast, possibly Anabaena). Receptors and other components of signal transduction systems allow cells to respond appropriately to their sisters or their neighbors (budding yeast, possibly fission yeast, and nematode ABa and ABp cells). Proteins in the cytoskeletal apparatus are responsible for determining the location and plane of cell division as well as the distribution of cytoplasmic determinants (nematode AB and PI cells, possibly Volvox). Other proteins function in marking DNA strands (fission yeast). Extension of the studies described above, as well as others not considered here because of space and thematic constraints, should provide further insights into the biochemical functions of these and other proteins that act in asymmetric cell divisions. More generally, the findings from each experimental system should help in the analysis of many of the others. The mechanisms that control asymmetric cell division in B. subtilis and in budding yeast have been presented in some detail because they exemplify the fact that none of the genes involved is acting in isolation; rather, each functions within an extensive regulatory cascade. Thus, as asymmetric cell divisions are further analyzed in multicellular organisms, it must be anticipated that the genes responsible will prove to encode components of pathways or possibly even of intricate networks of interacting genes. Indeed, multiple genes affecting single asymmetric cell divisions have already been identified in both nematodes (/in-17, /in-77, /in-78) and flies (numb, oversensitive). To understand how any gene functions in the control of asymmetric cell division, it will be necessary to elucidate both how it acts and how it is regulated. Genes that act in such pathways might be distinguished on the basis of whether they function in differentiation or in the generation of asymmetry per se. Consider again the cell division depicted in Figure 1, in which a cell divides to make sisters of different types A and B. If a mutation that results in a loss of gene function causes that division to generate two cells of type B, the gene defined by that mutation must be necessary for the sister cells to express different fates. Such a gene could act either to cause the asymmetry of the division or to effect that asymmetry once it has been specified. A gene required for expression of the differentiated B fate-whether or not a cell expressing that fate was generated by an asymmetric cell division-
would be of the latter class. myoD provides a plausible example of such a differentiation factor (Weintraub et al., 1989). Many of the genes discussed above could function similarly. By contrast, the C. elegans gene lin-77 affects the fates of a variety of distinct cell types and in each case acts to make sister cells different from each other. Thus, /in-l 7 seems likely to control the specification of the asymmetry of these cell divisions. Only by comprehensive analyses of both single genes and complete pathways of genes will both the determinants and the executors of asymmetric cell division be revealed. Acknowledgments We are deeply indebted to Erik Jorgensen for preparing the diagrams at the last moment. We thank Alan Grossman, Bob Haselkorn, Yuh Nung Jan, Ken Kemphues, David Kirk, Amar Klar, Rich Losick, Austin Newton, Lucy Shapiro, and membersof the Horvitz laboratory for many helpful suggestions concerning this manuscript. H. Fl. H. is an Investigator of the Howard Hughes Medical Institute.
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H., and Schnabel, R. (1987). The g/p-l locus in early C. elegans embryos. Cell 57, 601-
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Sulston, J. E., and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode Ceenorhebditis e&ens. Dev. Biol. 56, 110-156. Sulston, J. E., and White, J. G. (1980). Regulation and cell autonomy during postembryonic development of Ceenorhebdifis elegens. Dev. Biol. 78, 577-597. Sulston, J. E., Schierenberg, E., White, J. G., and Thomason, J. N. (1983). The embryonic cell lineage of the nematode Ceenorhebditis elegens. Dev. Biol. 100, 64-l 19. Sulston, J. E., Horvitz, H. R., and Kimble, J. (1988). Part A: Lineage Charts; Part B: Positions of Nuclei. The Nematode Ceenorhebditis elegens, W. Wood, ed. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 457-489. Sun, D., Stragier, P., and Setlow, P. (1989). Identification of a new sigma factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis. Genes Dev. 3, 141-149.
StJohnston, D., Beuchle, D., and Ntisslein-Volhard, C. (1991). steufen, a gene required to localize maternal RNAs in the Drosophila egg. Cell 66, 51-63.
Swoboda, K. K., Dow, Ceulobecter crescentus
Schierenberg, E. (1985). Cell determination during early embryogenesis of the nematode Ceenorhabditis elegens. Cold Spring Harbor Symp. Quant. Biol. 50, 59-68.
Tkacz, J., and Lampen, J. (1972). Wall replication in Seccheromyces species: use of fluorescein-conjugated concanavalin A to reveal the site of mannan insertion. J. Gen. Microbial. 72, 243-247.
Schmidt, R., Margolis, P., Duncan, Jr., and Losick, R. (1990). Control of sigma-F by sporulation regulatory BeciNus subtiffs. Proc. Natl. Acad.
Uemura, T., Shepherd, S., Ackerman, L., Jan, L. Y., and Jan, Y. N. (1989). numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58, 349-360.
L., Coppolecchia, R., Moran, C., developmental transcription factor proteins SpollAA and SpollAB in Sci. USA 87, 9221-9225.
Wang,
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C. S., and Vitkovic, L. (1982). Nucleoids CB15. J. Gen. Microbial. 126, 279-289.
D. (1991).
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dependent DNA-binding activity by stimutatory Proc. Natl. Acad. Sci. USA 87, 9761-9765.
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Weintraub, H., Tapscott, S. J., Davis, Ft. L., Thayer, M. J., Adam, M. A., Lassar, A. 8.. and Miller, A. D. (1989). Activation of musclespecific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. USA 885434-5438. Whitman, 18, 215.
C. (1876).
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Wilcox, M., Mitchison, G. J., and Smith, R. J. (1973). Pattern formation in the blue-green algaAn&aena. I. Basic mechanisms. J. Cell Sci. 72, 707-723. Wilcox, M., Mitchison, G. J., and Smith, Ft. J. (1975). Spatial control of differentiation in the blue-green algaAnahaena. Microbiology, 453463. Yochem, J., and Greenwald, I. (1989). g/p-l and /in-72, genes implicated in distinct cell-cell interactions in C. elegans, encode similar transmembrane proteins. Cell 58, 553-563. Zheng, L., and Losick, FL (1990). Cascade regulation of spore gene expression in Be&//us subtilis. J. Mol. Biol. 272, 645-660.
coat
January
24, 1992, Copyright
0 1992 by Cell Press
Mechanisms of Asymmetric Cell Division: Two Bs or Not Two Bs, That Is the Question H. Robert Horvitz” and Ira Herskowitzt *Howard Hughes Medical Institute Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 tDepartment of Biochemistry and Biophysics University of California, San Francisco San Francisco. California 94143
How sister cells can have different fates is a fundamental aspect of the problem of how cell diversity is generated during development. In principle, sister cells that aredifferent could be produced in two distinct ways. First, polar mother cells could divide to generate daughters that are different from the time they are formed. Alternatively, two identical sister cells could be generated and become different as a consequence of some later event. Consideration of these mechanisms raises immediate further questions: What causes a mother cell to be polar? How do initially identical sister cells become different? And in each case, how do initial differences in sister cells lead to their ultimately distinct fates? Answers to these questions would provide important insights into the mechanisms responsible for the control of development. In this review, we use the term “asymmetric cell division” to refer to any cell division in which sister cells have different fates. These distinct fates can be recognized by differences in size, shape, or in other morphological or biochemical features, or, if the sister cells are blast cells, by differences in their subsequent patterns of cell divisions and in the number and nature of the descendant cells they produce. Thus, if a cell division generates sister cells with distinct fates “A” and “B,” that division is considered to be asymmetric (Figure 1). By contrast, if a division generates sister cells with the same fate, e.g., 8, that division is symmetric. In short, the problem of asymmetric cell division is how a cell can divide to produce cells with distinct fates A and B instead of two cells that express the same fate B. As we use the term, an asymmetric cell division need not produce sister cells of different sizes and can produce initially identical cells with fates tllat differ as a consequence of subsequent cell interactions. Our current understanding of the mechanisms responsible for asymmetric cell division derives from a wide variety of experimental approaches using many different organisms. For example, classical observations of animal embryology established that specific regions of egg cytoplasm are partitioned to specific descendant cells that have distinct fates and also that certain substances are localized within the cytoplasm of the egg and can be differentially distributed to daughter cells. More recent studies involving micromanipulation or using a microelectrode to kill specific cells have revealed that cell interactions can cause sister cells to express different fates. Genes that
Review
are functionally responsible for asymmetric cell division have been identified in genetically tractable animals, such as nematodes and fruit flies, and the characterization of these genes has revealed aspects of the mechanisms by which they act. The most detailed studies of the mechanisms of asymmetric cell division have used easily studied unicellular organisms, including bacteria, yeast, and algae. Together these approaches are revealing the mechanisms responsible for specific asymmetric cell divisions and the degree to which these mechanisms are shared among organisms. We describe in this review examples of the two basic mechanisms noted above by which sister cells can become different, focusing on experimental systems in which the process can be studied at the level of single cells. We consider genetic analyses that have identified mutants in which the asymmetry of specific cell divisions is disrupted and what is known about the molecular mechanisms of action of the genes defined by these mutants. Finally, we discuss the similarities and differences among the mechanisms of asymmetric cell division that have been revealed by these various studies. Intrinsically
Asymmetric
Cell Divisions
The hypothesis that a cell division can produce two daughters that are intrinsically different from the time they are generated was formulated more than a century ago based upon studies of leech cell lineage, which revealed that distinct cytoplasmic domains of the leech egg are differentially partitioned to its descendants (Whitman, 1878). Subsequent observations of ascidian cell lineage supported this idea by identifying five distinct pigmented areas of egg cytoplasm that segregate to cells generating five distinct tissue types (Conklin, 1905). Such classical findings have been complemented by many modern studies in which specific molecules have been identified that are localized within specific regions of eggs and early blastomeres and that are distributed to specific descendant cells. Collectively, these observations have provided the basis for a fundamsntal hypothesis of developmental biology: specific molecules localized within regions of the cytoplasm of a mother cell can be distributed unequally to its two daughter cells and can act as developmental determinants to cause these daughters to express distinct characteristics and have different fates (Figure 2a). A comprehensive and stimulating review of the concept of cytoplasmic localization has been published by Davidson (1988). Mechanisms responsible for cytoplasmic localization of developmentally important molecules seem likely to be revealed by studies of Drosophila melanogaster genes that have RNA and/or protein products localized within tht egg (Driever and Niisslein-Volhard, 1988; Kim-Ha et al., 1991; Ephrussi et al., 1991; St Johnston et al., 1991). Although cytoplasmic localization provides an appealing mechanistic basis for intrinsically determined asymmetric cell division, other mechanisms can be imagined.
Cell 238
a
INTRINSIC
DIVERSITY
a
0
A
b
B
043 rc
00 + A 60
B db b
NO DIVERSITY
I
el
B Figure
1. Generation
of Diversity
by Asymmetric
(a) Asymmetric cell division generates (see text), labeled A and f3. (b) Symmetric cell division generates
L
LOCALIZED DETERMINANTS
B
ASYMMETRIC DIVISION PLANE
B CELL
Cell Division
sister cells of two distinct
SIGNALING
types
d
sister cells of the same type, B.
Any molecule that is asymmetrically segregated could in principle be used to distinguish sister cells and hence to serve as a developmental determinant. Molecules localized to the cell cortex (as opposed to regions of the cytoplasm), to one of the two centrioles, or to one of the two DNA strands of a chromosome, or even those DNA strands themselves all are candidates of this sort. Furthermore, intrinsic asymmetry need not be caused by an asymmetrically segregated molecule. For example, the asymmetric placement of the mitotic spindle would result in the generation of daughter cells of different sizes, which could in turn cause the expression of different fates (Figure 2b). Evidence for a number of these mechanisms exists, as discussed below. Rigorous proof that a specific asymmetric cell division is intrinsically determined can be difficult to obtain and in many cases necessitates an understanding of the underlying mechanism. Even two sister cells that have been generated in isolation from other cells could become different as a consequence of signaling that occurs between the sisters. The existence of an asymmetrically segregated molecule or a difference in cell size is not sufficient to establish that differences in sister cell fates are intrinsically determined. Such differences must be shown to cause the sister cells to be different. For these reasons, many examples of apparent intrinsic determination rely on circumstantial evidence, such as the polarity of the mother cell, the inequality of the size of the daughter cells, or the asymmetric segregation of specific molecules. We discuss below some selected examples in which the intrinsic nature of the determination of an asymmetric cell division seems likely and in which the mechanistic basis of this asymmetry could well be revealed in the near future. Caulobacter The bacterium Caulobacter crescentus can exist as either of two distinct differentiated cell types (reviewed by Newton and Ohta, 1990; Gober and Shapiro, 1991). Swarmer cells are motile, contain elaborate flagella, and are chemo-
0
WITH OTHER CELLS
Figure
2. Mechanisms
of Asymmetric
Cell Division
(a and b) Intrinsic mechanisms. (a) Localized developmental determinants responsible for fates A and B are differentially segregated to the two daughter cells. (b) Asymmetry in the division plane results in daughters of different sizes, which can cause them to express the different fates A and B. (c and d) Mechanisms dependent upon cell signaling. Initially identical sister cells of bipotential fate AIB interact with each other or with other cells to become different and express fates A or B. Such cells are said to constitute sister cell equivalence groups (see text). (c) Initially identical sister cells become different as a consequence of interactions (indicated by the dashed line) with another cell or cells (indicated by the square). (d) Initially identical sister cells at random become biased toward one fate or the other and then reinforce that bias via interactions (dashed line) between the sister cells.
tactic. Stalked cells are nonmotile and can attach to the substratum through a stalk. Swarmer and stalked cells differ in many ways, including in their patterns of gene and protein expression. In addition, stalked cells can initiate DNA replication and divide, whereas swarmer cells can do so only after losing their flagella and becoming stalked cells. Stalked cells divide to produce one stalked cell and one swarmer cell (Figure 3a). What determines the biochemical and morphological differences between the sister cells produced by this asymmetric cell division? The swarmer cell contains a variety of swarmer cell-specific proteins, including the chemotaxis receptor encoded by the mcpA gene, the flagellin encoded by the flgK gene, and the hook protein encoded by the flaK gene (Gomes and Shapiro, 1984; Ohta et al., 1985; Minnich and Newton, 1987). Both these proteins and the mRNAs that encode them are prin-
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Mechanisms
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(a)
Cell Division
SWARMER
-0 STALKED
(b)
Figure
CELL
}
DIFFERENT TRANSCRlPTlON
(c) pleC
CELL
FACTORS
DIFFERENT CHROMOSOMES
mutants
3. Caulobacter
(a) Only the nonmotile, stalked cell form of the wild-type bacterium Caulobacter crescentus can undergo cell division (Newton and Ohta, 1990; Gober and Shapiro, 1991). Within the predivisional cell, DNA replication occurs, and a flagellum is formed at the pole opposite to that at which the stalk is located. One daughter cell, like its mother, is a stalked cell, while the other is a motile and chemotactic swarmer cell. The swarmer cell can lose its flagellum, form a stalk, and then initiate cell division. (b) Models for how the pre-stalked and the pre-swarmer cell compartments of the predivisional cell differ in gene expression (see text). One possibility, shown on the left, is that these two compartments differ in their transcription factors (closed and open circles). Distinct proteins could be present or the same proteins could be differently modified causing them to be differentially active. A second possibility, shown on the right, is that the chromosomes in these two compartments are differentially active, perhaps as a consequence of the more condensed nature of the chromosome in the pre-swarmer cell compartment. (c) Mutants defective in the p/eC gene undergo a symmetric cell division, producing two progeny with defective flagella.
cipally synthesized in the predivisional cell (Gomes and Shapiro, 1984; Ohta et al., 1985), in which the flagellar apparatus assembles prior to cell division. After cell division, the swarmer cell continues to produce the f/gKflagellin (Minnich and Newton, 1987) apparently from mRNA synthesized in the predivisional cell and subsequently segregated to only the swarmer daughter cell (Milhausen and Agabian, 1983; Goberet al., 1991).Thisnewlysynthesized flagellin allows the flagellar filament to continue to grow.
These observations raise a number of questions concerning the asymmetric division that generates the swarmer and stalked cells. For example, what causes swarmer cellspecific proteins to be localized to the incipient swarmer cell pole of the predivisional cell? The answer, at least in part, is that some of these proteins, including the f/gKencoded flagellin, appear to contain sequences within them responsible for this localization (see Gober and Shapiro, 1991;Alleyet al., 1991). Theseproteinsmight associate with a site at the incipient swarmer cell pole (possibly a remnant from a prior cell division) that provides a morphogenetic landmark for assembling the complex flagellar and basal body structure. A second question also must be answered: what causes mRNA synthesized in the predivisional cell to be segregated specifically to the swarmer cell daughter? Localized transcription and the prevention of free diffusion within the predivisional cell seem to be responsible. Specifically, the late predivisional cell appears to consist of two compartments between which proteins and presumably other macromolecules do not freely exchange (Gober et al., 1991). Each of these compartments contains one of the two sister chromosomes present after replication but before division. Thus, if only one of these chromosomes expresses a particular complement of genes, the mRNA and protein products of those genes would be found in the corresponding subcellular compartment and hence in the corresponding daughter cell type. Support for such a mechanism has been provided by the observation that fusing the upstream regulatory region of the hook operon (which contains the f/aK gene) to a promoterless neomycin phosphotransferase reporter gene results in neomycin phosphotransferase reporter protein and translatable mRNA being found specifically in swarmer cells (Gober et al., 1991). The presence of the reporter protein in the swarmer cell is thought to reflect the localization of the expression of this gene within the predivisional cell. Thus, one aspect of the control of the asymmetry of Caulobacter cell division reduces to the question of why two chromosomes within the mother cell are differentially active. A number of mechanisms seem plausible (Figure 3b). For example, the compartment at the pre-swarmer pole might contain transcription factors that differ from those at the pre-stalked pole. Alternatively, the two chromosomes might exist in different states, resulting in differing affinities for specific transcription factors and/or in differing abilities to transcribe particular genes. Evidence that the nucleoids of stalked and swarmer cells have different sedimentation properties is consistent with this latter hypothesis (Evinger and Agabian, 1979; Swoboda et al., 1982). In either case, the question would simply be moved one step back, to the issue of what makes these differences between the two subcellular compartments. Thus, asymmetric cell division by Caulobacter involves a morphologically and molecularly polar mother cell for which it is known that aspects of this polarity causally result in the differences between the two daughter cells produced. To understand the mechanisms responsible for conferring this functionally important polarity to the mother cell, the molecules involved must be identified and charac-
Cdl 240
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. I...
-~~~~~~~~~
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membranes
4. B. subtilis
Figure based on Losick and Stragier (1991). (a) During spore formation of the bacterium B. subtilis, a vegetative cell divides asymmetrically to generate daughters of different sizes. The larger cell, known as the mother cell, engulfs the smaller cell, the forespore, which differentiates into the spore. The dashed line represents the cell wall that surrounds both daughter cells. (b) Differences in gene expression between the mother cell and the forespore result from differences in transcription factors. The oK transcription factor specifies the expression of mother cell-specific genes, and the oG transcription factor specifies the expression of foresporespecific genes. Expression of the gene that encodes oK is specified by another o factor, oE. Expression of the gene that encodes oG is specified by 19. Both oE and oF appear to be activated after the time of cell division, by the products of the .?pd/GA and spa//AA genes, respectively. (c)Cell signaling plays an important role during mother cell and spore differentiation. For example, oF activity in the forespore regulates oE activity in the mother cell; oE activity in the mother cell regulates o0 activity in the forespore; and aG activity in the forespore regulates oK activity in the mother cell.
terized. Mutations that affect the asymmetry of CaUlObatter cell division should help identify such molecules. For example, mutants defective in the gene p/eC (pleiotropit) form inactive flagella and no stalk, producing two morphologically identical daughter cells, each with a straight flagellum (Figure3c)(Sommer and Newton, 1989). Further characterization of p/eC has revealed that this gene interacts functionally with genes necessary for cell division, thereby linking the mechanisms responsible for cell division and those responsible for the generation of asymmetry (Sommer and Newton, 1991).
Bacillus subtilis When starved for nutrients, cells of the bacterium B. subtilis generate spores, which can survive harsh environmental conditions (Setlow, 1988; Kunkel, 1991). Spore formation involves asymmetric cell division (Figure 4a). A vegetative cell replicates its DNA to generate two daughter chromosomes that become separated by a septum that develops near one pole of the cell. One of the resulting compartments, called theforespore, differentiates into the mature spore. The second compartment, referred to as the mother cell, envelops the forespore and produces a protective coat that encases the spore. Mother cells are approximately four times the volume of forespore cells. Thus, during spore formation a B. subtilis cell divides to generate sister cells of two distinct types. These sister cells differ not only morphologically but also in the proteins they contain (Setlow, 1988; Zheng and Losick, 1990). In many cases these differences in proteins reflect differences in patterns of transcription, which in turn result from the effects of compartment-specific transcription factors. The genes responsible for the differences in mother cell and forespore gene expression have been analyzed in some detail (Figure 4b). For example, midway through differentiation, mother cells and forespores contain distinct transcriptional regulators: the mother cell contains a factor (oK) that recognizes promoters for mother cell-specific genes (Kunkel et al., 1989), and the forespore contains a factor ((I~) that recognizes promoters for forespore-specific genes (Sun et al., 1989). This difference in CJfactors is caused by differences in yet other o factors (reviewed by Stragier and Losick, 1990; Losick and Stragier, 1992). Thus, transcription of the gene that encodes the forespore-specific factor oG is specified by oF, which acts within the newly formed forespore (Margolis et al., 1991). Similarly, transcription of the gene that encodes the mother cell-specific factor oK is specified by another o factor, uE, which like aF acts only after the time of septum formation (Stragier et al., 1988). Interestingly, the genes that encode oF and oE are expressed in the predivisional vegetative cell, although these o factors are active only after cell division has occurred, and each is active in only one of the two sister cells. How are oF and oE activated in this cell type-specific way? The activation of oE involves proteolytic cleavage, since oE is synthesized as an inactive proprotein, the cleavage of which is controlled by the product of the spa//GA gene. The activation of oF involves two other genes, spollAA and spo//AB (Schmidt et al., 1990). These genes act in a negative regulatory cascade, such that the SpollAB protein negatively regulates the activity of oF and the SpoIlA!, protein negatively regulates the activity of the SpollAB protein. Thus, cell type-specific activity of spa//GA and spa//AA could lead to corresponding cell type-specific activity of uE and oF, respectively. Both the spa//GA and the spol/AA genes are expressed in the predivisional cell (Gholamhoseinian and Piggot, 1989). Margolis et al. (1991) have suggested that the SpollAA protein might be inactive prior to septation and activated within the forespore. If so, the question then becomes how SpollAA (and possibly SpollGA) activity changes in this way.
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Mechanisms
of Asymmetric
Cell Division
Gene expression in both the forespore and the mother cell is regulated by signaling between these two sister cells (Figure 4c) (reviewed by Losick and Stragier, 1992). Specifically, oF activity in the forespore is necessary for the proteolytic activation of aE in the mother cell; aE activity in the mother cell is necessary for the process of engulfment, which causes oG activation in the forespore; and oG activity in the forespore is necessary for the activation of oK in the mother cell. (oK activation, like oE activation, involves proteolysis of a proprotein.) This series of signals back and forth between the forespore and the mother cell has been named “crisscross regulation” by Losick and Stragier (1992) and provides a striking example of sister cells communicating with each other during the course of their differentiation. Thus, asymmetriccell division during B. subtilissporulation results in sister cells that at the time of their formation are different in size and probably different in some key molecular features, such as the activity of oF. For this reason, the asymmetry of this cell division can be considered to be intrinsically determined. Nonetheless, the sister cells generated then interact via cell signaling to drive further differentiation by activating a cascade of transcription factors responsible for cell type-specific gene expression. One step along the pathway to mother cell differentiation involves a genomic rearrangement: an approximately 42 kt region is excised in the mother cell to generate a fusion gene that encodes the oK protein (Kunkel et al., 1990). This rearrangement contributes to, but is not necessary for, the restriction of oK activity to mother cells. Budding Yeast Each cell division of the budding yeast Saccharomyces cerevisiae produces sister cells that are morphologically and functionally different (Figure 5a) (Strathern and Herskowitz, 1979; Nasmyth, 1983). These cells differ in size (Hartwell and Unger, 1977). The larger is referred to as the mother cell, and the smaller as the daughter cell. The cell surface of the daughter cell, or bud, is entirely newly synthesized (Tkacz and Lampen, 1972). Because the daughter cell must increase in size before it can initiate cell division, the two sister cells differ in their abilities to divide (Brewer et al., 1984). They also differ in their abilities to switch from one mating type to another, a process known as mating-type interconversion. Haploid budding yeast cells are of two mating types, a and a. An a cell can change its genotypic mating type to a, and an a cell can change to a. This mating-type interconversion requires the activity of the HO gene, which encodes an endonuclease that catalyzes a genomic rearrangement at the mating-type locus and causes interconversion between the a and the a states (Strathern et al., 1982). The HO product is thus a regulator of the mating-type locus, which is itself the master regulator of cell type in yeast (Herskowitz, 1989). Mother cells, but not daughter cells, can undergo mating-type interconversion (Strathern and Herskowitz, 1979). Specifically, mother cells are competent to divide to produce cells with switched mating type in the next generation, whereas daughter cells are not (Figure 5b).
This difference between the two sister cells produced by each cell division is propagated through the yeast cell lineage. Yeast sister cells differ in their abilities to undergo mating-type interconversion because only mother cells can transcribe the HO gene (Nasmyth, 1983); if daughter cells are caused to transcribe the HOgene, these cells also can switch mating type (Jensen and Herskowitz, 1984). Transcription of the HO gene is regulated by more than a dozen regulatory proteins (reviewed by Herskowitz, 1989). Six genes, SW/l to SW16, are required for transcription of HO. Five other genes, SIN1 to SIN5, were identified because inactivation of these genes relieves the requirement for various SWI genes. Thus, these SIN genes behave as if their products negatively regulate HO transcription. Mutations in the SWI and SIN genes cause the normally asymmetric division of budding yeast to be symmetric instead (Figure 5b) (Nasmyth et al., 1987a; Sternberg et al., 1987; Kruger, 1991). Specifically, mutantsdefective in any of the SWI genes produce sister cells that are identical in the sense that neither cell can switch mating type (nonswitchable, NS). By contrast, mutantsdefective in the S/N7 or the SIN3 genes (the only SIN genes tested so far) also produce sister cells that are identical, in this case with both sisters being able to exhibit switching (switchable, S). Thus, the SWI and SIN genes are necessary for the asymmetry of budding yeast cell division with respect to competence to undergo mating-type interconversion. Gene interaction studies have indicated that the SWI and SIN genes act in a negative regulatory cascade in the order: SW15 + SIN3,4 -j SWll,2,3 + SIN1,2 + SWl4,6-, HO (Herskowitz, 1989). The products of most of the SWI and SIN genes have been characterized molecularly, leading to the following model. The SW14 and SW16 proteins, which cause HO gene expression, are transcription factors specific for genes expressed in the Gl phase of the cell cycle (such as HO) (Breeden and Nasmyth, 1987; Nasmyth and Dirick, 1991; Ogas et al., 1991). The SIN1 and SIN2 proteins are chromatin components that are thought to antagonize the activities of the SW14 and SW16 proteins (Kruger and Herskowitz, 1991; Kruger, 1991). The SWIl, SWl2, and SW13 proteins are general transcription factors that antagonize the activities of SIN1 and SIN2 (Peterson et al., 1991; unpublished data). The SW15 protein is a zinc finger transcription factor (Nagai et al., 1988) that is thought to antagonize the activities of the SIN3 and SIN4 genes, which in turn are thought to antagonize the activities of the SWII,SWl2, andSW/3genes(Herskowitz, 1989).Thismodel IS depicted in Figure 5c. How do the activities of the SWI and SIN genes lead to HOtranscription in only mother cells? One hint is provided by the observation that if SW15 protein is ectopically produced in daughter cells, daughter cells can switch mating types (Nasmyth et al., 1987b). Thus, not only is SW15 protein needed for switchtng by mother cells, but its absence normally prevents switching by daughter cells. This observation has focused attention on the question of why mother cells but not daughter cells contain functional SW15 protein. Some possibilities derive from the relative timing of SW15 protein synthesis and function.
Cell 242
a
b
0
MOTHER CELL
SIN-
0
DAUGHTER CELL Figure
(,I,,., ) -x+ HO
SW15 binds and antagonizes R
(,lNl., ) -x+ HO
SWll,2,3 antagonize
SlNl,2
HO
SW14,6 bind and trigger HO transcription and swltchlng
HO
SW15 must act during the Gl phase of the cell cyclethe time when HO transcription occurs (Nasmyth, 1985). Newly synthesized SW15 protein enters the nucleus just prior to Gl phase (Nasmyth et al., 1990; Moll et al., 1991). However, the SW15 gene is transcribed during late S phase, substantially later in the cell cycle (Nasmyth et al., 1987b). Thus, a cell’s functional complement of SW15 protein must be synthesized during the preceding cell cycle. These considerations suggest that SW15 protein synthesized in a dividing cell might be preferentially segregated to its mother cell rather than to its daughter cell. However, immunocytochemical studies of the localization of the SW15 protein have revealed its presence in the nuclei of both of these sister cells (Nasmyth et al., 1990). Some
5. Budding
Yeast
(a) The budding yeast S. cerevisiae divides to generate two unequally sized sister ceils. The smaller, known as the daughter cell, develops from a bud formed from the parent cell. The larger of the two sister cells is known as the mother cell. (b)Thefatesof thetwosister cellsgenerated bythe division of wild-type budding yeast can be distinguished based upon their abilities to undergo mating-type interconversion. The mother cell is competent to switch (S) (i.e., to generate progeny of the opposite mating type), whereas the daughter cell is not competent to switch (NS). In SWIt mutants, neither of the progeny cells can switch, whereas in SINmutants both of the progeny cells can switch. (c)Switching in budding yeast is driven by an endonuclease, the product of the HO gene, which makes a double-strand break at the matingtype locus. Expression of the HO gene is regulated by a cascade of proteins. One model for the action of these proteins is as follows. HO gene expression is triggered by the SW15 transcription factor, which binds to the upstream regulatory region of the HO gene and is hypothesized to displace a negative regulator (R), the function of which depends upon the SIN3 protein (Wang and Stillman, 1991). Based upon gene interaction experiments, the SIN4 protein acts at a similar step, but its mechanism of action is unknown. The SWIl, SWl2. and SW13 general transcriptional regulators are thought to be facilitated by the SWIB-DNA complex and thereby antagonize the SIN1 and SIN2 proteins, both of which are components of chromatin. With these proteins gone, the SW14 and SW16 proteins, which are transcriptional regulators that function to induce gene expression during the Gl phase of the cell cycle, can bind and activate HO transcription. In this way, SW15 activity leads to HO gene expression and to mating-type interconversion.
other mechanism must be responsible for the difference in SW15 function between mother and daughter cells. One model that has been suggested is based upon the size difference between the two sister cells (Sternberg et al., 1987; Nasmyth et al., 1990). The smaller daughter cell must increase substantially in size before it can reach the stage of the cell cycle at which HO transcription can begin (Brewer et al., 1984). Perhaps the SW15 protein segregated to both mother and daughter cells is unstable, and the longer delay in the smaller daughter cells results in the degradation of SW15 protein to an extent that HO transcription cannot take place (an idea challenged by Nasmyth et al. (1990)). Alternative models are also possible, and the basis of the differing levels of SW15 activity between mother and daughter cells remains a major issue. Thus, the difference between the abilities of budding
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Mechanisms
a
of Asymmetric
Cell Division
b
an
0 d
\
nn
FISSION
YEAST Figure
C
S
NS
d
yeast sister cells to undergo mating-type interconversion is controlled by a negative regulatory cascade of proteins that control the expression of an endonuclease that specifies cell type. Current data indicate that the SW15 protein acts at the earliest known point in this cascade, and it seems likely that differences in SW15 activity between mother and daughter cells are conferred intrinsically, at the time of cell division. Nonetheless, later stages of the differentiation of the a and a cell types involve cell interactions, as each mating type produces a diffusible sexual pheromone that influences the differentiation of the other (reviewed by Herskowitz, 1989). This two-step process of making sister cells intrinsically different and then reinforcing those differences by cell interactions between these cells is similar to that used for the control of asymmetric cell division by B. subtilis, as described above. Fission Yeast Each cell division of the fission yeast Schizosaccharomyces pombe produces sister cells that appear to be morphologically identical. Nonetheless, these cells have dif-
6. Fission
Yeast
(a) The fission yeast S. pombe divides to generate one daughter cell that is competent to undergo mating-type interconversion (S) and one daughter cell that is not competent (NS) (Miyata and Miyata, 1981). (b) A fission yeast cell that switches generates one daughter of a different mating type and one daughter of the same mating type as its parent cell (Miyata and Miyata, 1981). For example, this diagram shows a cell of mating type M dividing to produce one S and one NS cell. The S cell, also of mating type M, produces one cell of mating type M and one cell of switched mating type P. The NS cell produces two cells of mating type M. By contrast, a budding yeast cell that switches generates two daughters that differ in mating type from their parent. For example, a cell of mating type a divides to produce one S and one NS cell. The S cell, also of mating type a, produces two cells of mating type a. The NS cell produces two cells of mating type a. (c) Model for how DNA strand modification can confer differences between sister cells (modified from Klar, 1987). A parent cell with one DNA strand modified (solid ellipse) at the mating-type locus (arrows) will produce one daughter (on the left) with this modified DNA strand and one daughter (on the right) in which the newly synthesized corresponding DNA strand is unmodified. Only the daughter cell with the modified DNA strand is competent to undergo mating-type interconversion. The solid and open arrows represent the two complementary DNA strands. The thick and thin arrows represent old and new DNA strands, respectively. (d) An inverted tandem duplication of the mating-type locus allows both DNA strands to be modified and hence both daughter cells to undergo mating-type interconversion (modified from Klar, 1990).
ferent fates, since they differ in their abilities to undergo the process of mating-type interconversion in their next cell division(Miyataand Miyata, 1981). Like budding yeast, fission yeast has two mating types, which in this case are named P (plus) and M (minus). P cells can generate M cells, and M cells can generate P cells This mating-type interconversion involves a genomic rearrangement that is in some respects mechanistically similar to that involved in mating-type interconversion in budding yeast (Egel et al., 1984). Of a pair of fission yeast sister cells, one can switch mating type (the S cell in Figure 8a) and the other cannot (the NS cell) (Miyata and Miyata, 1981). This phenomenon is similar to that seen in budding yeast, in which the mother cell, but not the daughter cell, can switch. There are two striking differences between fission yeast and budding yeast in mating-type interconversion. First, in fission yeast a cell that switches (S) generates cells of the two different mating types (Figure 8b). (A cell that does not switch [NS] generates two cells of the same mating type as the parent cell.) For example, an M cell that switches generates one M and one P cell. By contrast, in budding
Cell 244
yeast, a cell that switches generates two cells of the opposite mating type. Thus, an a cell that switches generates two a cells. These differences probably arise from differences in the time during the cell cycle when the genomic rearrangement occurs: if rearrangement precedes replication of the mating-type locus, both daughters should switch, as in budding yeast, whereas if rearrangement follows replication, only one daughter should switch, as in fission yeast. The second and more significant difference between fission and budding yeasts concerns the basis for the asymmetry of their divisions (Klar, 1987). As discussed above, budding yeast sister cells differ in their abilities to produce the endonuclease that initiates genomic rearrangement (Nasmyth, 1983; Jensen and Herskowitz, 1984). By contrast, fission yeast sister cells appear to differ in their substrates for genomic rearrangement, namely, in the chromosomes that carry that mating-type locus. This chromosomal difference was discovered by Egel (1984), who found in studies of diploid fission yeast cells that the two homologous chromosomes containing the matingtype locus could switch independently. This observation suggests that the difference in switching competence between sister cells is not conferred by cytoplasmic differences (e.g., in an enzyme such as an endonuclease), but rather by differences in the state of the mating-type locus itself. Subsequent studies have supported the hypothesis that the substrate for genomic rearrangement is different in the two sister cells produced by the division of fission yeast. Klar(1987) proposed that a modification of one DNAstrand (e.g., by methylation) occurs at the mating-type locus (Figure 8~). Because DNA replication is semiconservative, this marked strand will generate a marked daughter chromatid that differs from its sister chromatid, in which the corresponding DNA strand is newly synthesized and hence unmodified. The marked and unmarked chromatids will be segregated to the two daughter cells, causing those cells to be molecularly distinct and thereby allowing only one, for example that with the marked chromatid, to produce progeny that switch. The newly synthesized and unmodified strand in the other cell could be modified subsequently in the cell cycle, thereby allowing this cell, like its parent cell, to generate one daughter that produces progeny that switch and one that does not. This model predicts that if two copies of the mating-type locus are present in opposite orientations, a site for modification should exist on both strands and thus allow both daughters to produce progeny that can switch (Figure 6d). Indeed, Klar (1990) observed that an inverted tandem duplication at the mating-type locus causes sister cells that would otherwise be different to instead be the same in this way. Although it is appealing to think that the DNA modification that occurs at the mating-type locus is covalent, it remains possible that this modification is noncovalent, involving for example the binding of a protein specific for one DNA strand and available at only certain times of the cell cycle. The normally asymmetric cell division pattern of fission yeast can also be caused to be symmetric hy mutation. A
variety of swi genes (which bear no known relationship to the SW/genes in budding yeast) have been identified that are necessary for switching (Egel et al., 1984). Mutants in these genes produce sister cells, neither of which can undergo mating-type interconversion. In summary, asymmetric cell division by fission yeast appears to be a consequence of the asymmetry that results from semiconservative DNA replication. Fission yeast, like budding yeast, produces mating pheromones and other molecules that function in cell interactions during mating (reviewed by Marsh et al., 1991). Thus, it seems likely that fission yeast, like budding yeast (see above), uses signaling between cells of opposite mating type to lead to further differentiation of two sister cells that are intrinsically different. Nematode Of the 949 nongonadal cell divisions that occur during the development of the nematode Caenorhabditis elegans, 807 are asymmetric; that is, these divisions generate daughter cells that either differentiate in different ways or that divide to generate different sets of descendant cells (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979; Sulston et al., 1983, 1988). The C. elegans asymmetric cell division that has been analyzed in the most detail is the first cleavage of the egg. The asymmetry of this first cell division seems likely to be intrinsically determined (reviewed by Strome, 1989). We summarize below what is known about this cell division to exemplify the types of experimental approaches that have been taken. We choose this example from among many that have been studied using the methodologies of experimental embryology (e.g., Davidson, 1986) because it is being subjected to a genetic analysis that promises to reveal the molecules and mechanisms responsible for an intrinsically determined asymmetric cell division (Kemphues et al., 1988). The C. elegans egg divides to form two differently sized daughters, a larger anterior cell called AB and a smaller posterior cell called PI (Figure 7a). Thus, these sister cells are different at the time they are generated. AB and P, are clearly distinct in their fates. First, they undergo different patterns of cell division: AB displays a series of synchronous cleavages, each of which produces sister cells that are equal in size, whereas PI is a stem cell with descendants that are asynchronous in their times of division and unequal in their sizes. Second, AB and PI generate different cell and tissue types: AB produces primarily ectoderm, whereas PI produces mesoderm, endoderm, and the gonad, as well as some ectoderm. A number of observations indicate that AB and PI have distinct developmental potentials. First, studies of isolated AB and PI cells indicate that differences between the fates of these cells do not depend upon interactions either between them or among their descendants. Laufer et al. (1980) and Edgar and McGhee (1986) squashed eggs to lyse either AB or P,, and Priess and Thomson (1987) surgically removed either AB or PI using a microneedle. In the resulting partial embryos, an isolated AB cell generates equally sized daughters that divide synchronously to produce many descendants that become ectodermal cells, as
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(b) Model: cytoplasmic
Cell Division
determinants
(c) par mutants
for P,
(d) g/p-7 mutants
f Figure
7. Nematode
Embryonic
Cell Divisions
(a) The C. elegans egg divides asymmetrically to generate a larger anterior daughter AB and a smaller posterior daughter PI. AB and P, differ in many ways. AB then divides asymmetrically to form an anterior daughter ABa and a posterior daughter ABp, while P, divides asymmetrically to form an anterior daughter EMS and a posterior daughter Pz. ABa, ABp, EMS, and PI all differ in their fates. (b) One model for the role of cytoplasmic determinants in the first egg division. Determinants responsible for specifying the P, fate are concentrated within the posterior cytoplasm of the egg and segregated preferentially to the posterior daughter cell. (c) Mutations in the par genes cause the first zygotic cell division to be symmetric instead of asymmetric, generating two nearly identical sister cells that fail to produce certain PI descendants and can produce certain AB descendants in excess, suggesting that the two daughter cells are both AB-like in character. (d) Mutations in the g/p-l gene appear to cause the two daughters of AB to both express the ABp fate. (e) Two models for the role of the Glp-1 protein, which has been postulated to be a cell surface receptor, in the cell interactions responsible for making the AB cell divide asymmetrically. In both models, a signal from EMS causes the anterior AB daughter to express the ABa fate instead of theABpfate. In thefirst model,shownabove, theGlp-I protein isthereceptorfortheEMSsigna1. In thesecond model, shown below, aGlp-l-mediated signal from the posterior daughter acts in conjunction with a signal from EMS to elicit the ABa fate. In either case an absence of EMS signal or of Glp-1 receptor would cause the anterior daughter to express the ABp fate.
in the intact embryo. An isolated PI cell generates unequally sized daughters that divide asynchronously to produce descendants that twitch and contain intestinespecific markers, again as in the intact embryo. Similarly, after cytokinesis has been blocked using cytochalasin, a PI cell expresses muscle-specific and intestine-specific
markers, while A6 does not (Laufer et al., 1980; Cowan and McIntosh, 1985; Edgar and McGhee, 1988). Together, these studies establish that a number of the differences between the descendants of AB and those of P, are determined by the two-cell stage and do not depend upon interactions between AB and PI.
Cell 246
The difference in developmental potential between AB and PI appears at least in part to be conferred by a cytoplasmic factor (Figure 7b), since the cytoplasm of PI seems to be able to induce expression by AB of intestine-specific markers: embryos in which the P, nucleus was extruded through a hole made by laser microsurgery and in which the cytoplasms of AB and PI were fused displayed the autofluorescence characteristic of differentiated intestinal cells (Schierenberg, 1965). In addition, the potential of the egg to divide to produce two unequally sized daughters might be conferred by activity localized to its posterior cytoplasm, since extrusion of its posterior but not of its anterior cytoplasm has been reported to cause the egg to produce equally sized daughters (Schierenberg, 1965). Although the factor(s) responsible for conferring these differences between AB and P, has not been identified, cytoplasmic elements are known to be asymmetrically segregated during cleavage of the C. elegans egg, establishing that an appropriate mechanism exists for the segregation of developmental determinants. Specifically, P granules, which are found in germ cells at all developmental stages, are localized to the posterior pole of the egg and upon division segregate to P, but not to AB (Strome and Wood, 1962). Together these observations suggest that differences between AB and PI might well be conferred by the differential segregation of factors asymmetrically localized within the egg. What mechanisms are responsible for the localization of cytoplasmic factors within the nematode egg? During its first cell cycle, the fertilized egg undergoes a major cytoplasmic reorganization (Albertson, 1964) similar to that seen in many other embryos and believed to be responsible for cytoplasmic localization (Davidson, 1966). The treatment of C. elegans embryos with microfilament inhibitors (but not with microtubule inhibitors) disrupts this cytoplasmic reorganization, as well as P granule segregation and other indications of egg cell polarity, suggesting that actin microfilaments play a role in establishing this polarity (Strome and Wood, 1963; Hill and Strome, 1990). To identify other molecules that function in controlling cytoplasmic localization within the egg, Kemphues and coworkers (1966) have taken a genetic approach. Eight maternal effect lethal mutants defining four par (partitioning-defective) genes fail in P granule localization and other aspects of cytoplasmic reorganization (Kirby et al., 1990). These mutants in general produce AB and PI cells of similar sizes, which divide at the same time and continue dividing synchronously. Intestinal and germline cells, which are normally derived from PI, are missing, whereas muscle cells normally derived from AB are produced in excess in at least some par mutants. These and other observations have led to the suggestion that in par mutants the egg divides to form two nearly identical sister cells that both express an AB-like or a hybrid ABlP, fate (Figure 7~). A reasonable hypothesis is that the par genes function in cytoplasmic localization within the egg and are necessary for the determination of the PI cell fate. Further analysis of these and similar genes promises to identify the molecules responsible for these processes.
Asymmetric Ceil Divisions by Cell Signaling
Determined
How can two sister cells that are initially identical in their developmental potentials become different? First, one or both of the sister cells could respond to signals from other cells (see Figure 2~). Intercellular signaling is common in the development of many organisms (Gurdon, 1967; Pawson, 1991; Gurdon, 1992, this issue; Jesse11 and Melton, 1992, this issue), and signaling that influences 1 of 2 equipotent sister cells could act via mechanisms and molecules like those involved in other aspects of intercellular communication. Alternatively, stochastic events could cause two sister cells to differ in a small way that might be reinforced by a feedback mechanism involving signaling between the two sister cells (see Figure 2d). Many examples of sets of cells that are equivalent in developmental potential but that express different fates as a consequence of cell interactions are known. Such sets of cells are called “equivalence groups” and were first identified and have been most extensively characterized in C. elegans(Kimble et al., 1979; Sulston and White, 1960; Sulston, 1966; reviewed by Greenwald and Rubin, 1992, this issue). We discuss below examples of equivalence groups that consist of sister cells. Nematode The two daughters of the C. elegans AB blastomere constitute an equivalence group. The anterior daughter ABa and the posterior daughter ABp (Figure 7a) differ drastically in their fates, generating different numbers and types of cells and undergoing distinct patterns of cell division (Sulston et al., 1963). For example, ABa generates muscles of the pharynx, whereas ABp does not. Priess and Thomson (1967) used a micromanipulator to exchange the positions of ABa and ABp and found that the fates of these cells were then reversed. Normal and fertile animalsdeveloped, establishing the completeness of the transformation of the fates of these cells. This experiment revealed that each of these two cells when generated has the potential to express either the normal ABa or the normal ABp fate; which fate is expressed depends on cell position, presumably as a consequence of cell signaling. Priess and Thomson (1967) also found that after puncturing the eggshell and squeezing the EMS cell (Figure 7a) through the resulting hole, ABaderived pharyngeal muscles did not form. This observation suggests that EMS or its descendants induce ABa or its descendants to generate pharyngeal muscle, and that the different fates of the sister cells ABa and ABp are at least in part a consequence of interactions with another cell or cells. One molecule that functions in the determination of the ABa fate and is likely to act in cell signaling has been identified by genetic studies. Priess et al. (1967) isolated four mutants that fail to generate ABaderived pharyngeal muscle cells and that have other characteristics like those seen in embryos that lack the EMS cell (Figure 7d). All four mutants are abnormal in the same gene, g/p-l (germline proliferation defective), which encodes a member of a con-
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Figure based on Kuwada and Goodman (1985). (a) The asymmetric division of the ganglion mother cell GMC-1, a daughter of neuroblast NB l-1, generates sister cells that differentiate into two distinct neuron types, the anterior corner cell aCC and the posterior corner cell pCC. (b) The asymmetric division of the midline precursor cell MP3 generates sister cells that differentiate into two distinct neuron types, known as the H cell (because of its H-like shape) and the H cell sib.
served protein family that includes the products of the C. elegans gene lin-12 and of the Drosophila gene Notch (Yochem and Greenwald, 1989; Austin and Kimble, 1989; reviewed by Greenwald and Rubin, 1992). These proteins seem likely to act as cell surface receptors that function in intercellular signaling via direct cell contact (Kidd et al., 1989; Fehon et al., 1990). One possibility is that ABa is different from its sister ABp as a result of a signal from EMS that is received by Glp-1 protein located on the surface of ABa (Figure 7e). Alternatively, a signal from EMS and a parallel signal (possibly from ABp) received by Glp-1 protein located on the surface of ABa might be required together for the expression of the ABa fate. Grasshopper Two examples of sister cell equivalence groups were discovered during an analysis of the embryonic development of the grasshopper nervous system (Kuwada and Goodman, 1985). First, the two daughters of the ganglion mother cell GMC-1 differentiate into the morphologically clistinct anterior and posterior corner cells, known as the aCC and pCC neurons, respectively (Figure 8a). When one of these two daughter cells, chosen at random, was killed using an intracellular microelectrode, the remaining cell acquired characteristics of the pCC neuron in 8 of 9 cases. This experiment indicated that both cells can express the pCC fate and suggested that at the time they are formed, the two sister cells are equivalent in their developmental potentials. Similarly, the midline precursor cell MP3 divides asymmetrically to generate two morphologically distinct neurons, the H cell (which is shaped like the letter H) and the l-i cell sib (Figure 8b). When one of these two cells was killed using a microelectrode, the surviving cell generally expressed characteristics of the H cell sib. Thus, both of these cells appear to have the potential to express the H cell sib fate and thus are likely to constitute a sister cell equivalence group. Whether the more anterior or the more posterior of the two sisters becomes the H cell sib varies randomly, which supports the hypothesis that the cells are equivalent in their developmental potentials. The sister cells in each of these two cases must interact, since when both cells are present, only one expresses the
230 V
Figure
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9. Anabaena
(a) Vegetative (V) cells of at least one strain of Anabaena can divide asymmetrically to produce a larger daughter that differentiates into a vegetative cell and a smaller daughter that differentiates into a nitrogen-fixing heterocyst (H). (b) The asymmetry of Anabaena cell division can be influenced by intercellular signaling. A signal (dashed line) from a nearby heterocyst can cause a presumptive heterocyst to differentiate into a vegetative cell.
potential that it would express if the other cell were absent. In principle, this interaction could be either active or passive. For example, a cell expressing one fate could signal its sister to prevent it from expressing that fate. Alternatively, there could be a competition between the sister cells for proximity to (or distance from) the source of a signal that determines which of the two fates the cells will express. This latter mechanism is common in non-sister cell equivalence groups studied in C. elegans. For example, the ectoblast cells P5/8L and P5/8R constitute an equivalence group (reviewed by Horvitz and Sternberg, 1991). These two cells are generated on the left and right sides of the animal, respectively, and then centralize, so that one becomes located anterior to the other. Their subsequent fates depend on their anterior-posterior positions, as a consequence of their having assumed different positions with respect to a signal from a nearby cell, the anchor cell of the gonad. Cyanobacteria Cyanobacteridof the genus Anabaena grow as filaments of identical vegetative cells that divide about once a day (Wilcox et al., 1975). Heterocystous cyanobacteria respond to an environment with limiting nitrogen by generating terminallydifferentiatedcellscalled heterocysts, which function in nitrogen fixation and differ from vegetative cells in many morphological and biochemical respects. A new heterocyst will develop along a growing filament at a position approximately midway between adjacent preexisting heterocysts, maintaining a pattern in which heterocysts occur at approximately equal intervals along a filament. Since vegetative cells neighbor each heterocyst, asymmetric cell divisions cause certain vegetative cells to divide to generate one vegetative cell and one heterocyst (Figure 9a). What causes a heterocyst to become different from its vegetative cell sister? Vegetative cells of one strain of Anabaena divide to form sister cells of different sizes (Wilcox et al., 1975), and heterocysts develop from only the smaller of these cells. Since this difference in cell size is defined
Cdl 240
by the location of the septum in the mother cell, it would seem that this asymmetric cell division is intrinsically determined. However, not all of the smaller daughters differentiate into heterocysts. Specifically, only those small cells sufficiently distant from nearby heterocysts differentiate into heterocysts. Furthermore, if a developing heterocyst is punctured with a microneedle, a new heterocyst will form nearby (Wilcoxet al., 1973). These observations suggest that heterocyst formation is inhibited by signals from other heterocysts (Figure 9b). Since heterocysts occur periodically along a filament (about every five or six cells in the strain studied by Wilcox), this inhibitory signal can act at a distance. This role of cell signaling in asymmetric cell division contrasts with that seen in nematodes and grasshoppers, as described earlier in this section: in nematodes and grasshoppers cell signaling causes sister cells that are initially identical to become different, whereas in Anabaena cell signaling prevents sister cells that are initiallydifferent (at least in size) from becoming different cell types. Buikema and Haselkorn (1991a) have identified a mutant that fails to differentiate heterocysts from among a collection of mutants defective in nitrogen fixation. This mutant is defective in the hetR gene, which encodes a novel protein with no recognized sequence motifs (Buikema and Haselkorn, 1991 b). Overexpression of the herfi gene causes an increased frequency of heterocyst formation, even on media containing nitrogen. These observations indicate not only that hetR function is necessary for heterocyst formation, but also that the addition of hetR function is sufficient to cause cells that would not normally become heterocysts to do so. In other words, hetf? controls heterocyst formation. It seems likely that hetR regulates two DNA rearrangements that occur during heterocyst formation (Golden et al., 1985,1988), although thisregulation could be quite indirect. Since heterocysts can form in the absence of at least one of these rearrangements (Golden et al., 1988), these rearrangements could be consequences rather than causes of heterocyst differentiation. volvox Development of the spherical multicellular green alga VolVOX carteri forma nagariensis involves asymmetric cell divisions that produce cells of groksly differsnt sizes and strikingly different fates (reviev+ by Kirk et al., 1991). Whether the asym intrinsically or by cell i less, it is clear that t which cells ciivlde division, and hence the asymmetrically, is contr sexual hormones. Development of the asexual forni of Volvox carteri forma nagariensis begins with five symmetric cell divisions, which generate 32 cells of nearly equivalent size. The sixth cell division of the 18 anterior cells is asymmetric, generating a small anterior daughter and a large posterior daughter (Figure lOa). The 18 large cells divide asymmetrically twice more, in each case forming a small and a large daughter. The 16 terminal large daughters differentiate into the 16 asexual reproductive or gonidial cells of the
adult. These large cells are known as gonidial initials. All other cells produce only somatic cells, and are known as somatic initials. The somatic initials generate the total of about 2000 somatic cells found in the adult. The symmetry of specific cell divisions can be influenced by cell signaling (Figure lob). Although male and female strains are morphologically indistinguishable in their asexual phases, these strains are visibly dimorphic in their sexual phases. A pheromone produced by mature sexual males causes cells in the sixth round of cell division in developing female embryos to divide symmetrically instead of asymmetrically. Asymmetric divisions occur in the seventh round instead. Furthermore, more cells divide asymmetrically in such pheromone-treated females than in individuals not exposed to pheromone. The smaller cells are somatic initials, and the larger cells differentiate into eggs. Similarly, in sexually induced male embryos, asymmetric cell division is suppressed in the sixth, seventh, and possibly eighth rounds of cell division. Only the last round of cell division is asymmetric, generating equal numbers of somatic cells and precursor cells that divide symmetrically to produce packets of 64 or 128 sperm cells. During both normal asexual and sexual development as well as under a variety of abnormal conditions (e.g., after heat shock, subsequent to surgical manipulation, and in mutants), cell size and cell fate are strongly correlated, with larger cells invariably becoming reproductive cells. This observation has led to the hypothesis that it is the difference in the sizes of sister cells that is responsible for the difference in their fates (Pall, 1975; Kirk et al., 1991). Mutantsof Volvox have been isolated in which asymmetric cell divisions do not occur (Figure 10~). Because these mutants fail to generate gonidia, they are known as gonidia-less and define the gene g/s. Kirk et al. (1991) propose that the product of the gls gene functions in the placement of the Volvox mitotic apparatus and that when this gene is defective, the mitotic apparatus by default becomes located at the middle of the cell. Genes Required Cell Divisions
for the Asymmetry
of Specific
Genetics offers a powerful approach toward the elucidation of mechanisms that make sister cells different. Mutants in which sister cells that normally express different fates instead express the same fates can define genes that function in making sister cellsdifferentfrom each other. An analysis of such genes and their products should reveal the basis of their actions. For example, as discussed above, C. elegans mutants that cause the cell ABa to have characteristics of its sister cell ABp are altered in the Glp-1 protein, which is probably a cell surface receptor. This observation indicates that differences between ABa and ABp are most likely caused by cell signaling. A number of genes have been identified both in C. elegans and in Drosophila that are required for the asymmetry of specific cell divisions. Whether the sister cells generated by those divisions are different when they are formed or become different afterward is not yet known. The study of these genes should indicate not only whether or not
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these asymmetric cell divisions are intrinsically determined, but also should lead to an understanding of the complete genetic and biochemical pathways that are responsible for making these sister cells different. Nematode Studies of mutants abnormal in the development of the vulva of the C. elegans hermaphrodite have identified four genes necessary for the asymmetry of specific cell divisions (Ferguson et al., 1987). Mutations in each of these genes perturb the asymmetry of 1 of 2 sequential cell divisions involved in vulva1 development (Figure lla). The vulva is generated by three ventral ectodermal blast cells known as P5, P6, and P7. Each of these three P cells divides asymmetrically to form an anterior neuroblast (Pn.a cell, shown as NB in Figure lla) and a posterior ectoblast (Pn.p cell, shown as ECT). Two of the posterior ectoblasts (P5.p and P7.p) then divide asymmetrically to form two different classes of ectoblast cells (LL, TN), which are distinguished by both their subsequent division patterns and the numbers and types of descendant cells they generate. In /in-26 (lineage abnormal) mutants, the divisions of the P cells are symmetric instead of asymmetric: the two sister cells generated are both neuroblasts, apparently because each posterior daughter expresses the fate normally expressed by its sister (Figure 11 b) (Ferguson et al., 1987). Mutants defective in the gene In-1 7 have a similar effect
0 ++
10. Volvox
Figure based on Kirk et al. (1991). (a) During asexual development of individuals of either genotypic sex, asymmetric cell division commences with the sixth round of cell division and continues through the eighth round. The large daughter cells generated by the eighth round of cell division differentiate into the asexual reproductive cells of the adult. (b) The symmetry of cell division is influenced by a sexual pheromone (dashed fine), which is produced by sexually mature males and by heat-shocked asexual individuals. This pheromone delays the onset of asymmetric cell division to the seventh round in female zygotes and to the eighth or ninth round in male zygotes. The large daughter cells generated by the seventh round in female zygotes become eggs, and those generated by the eighth or ninth rounds in male zygotes become precursor cells that each generate 64 or 128 sperm. (c) In the g/s mutant, all cell divisions are symmetric in both asexually and sexually developing zygotes.
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on the subsequent asymmetric cell division: two of the ECT cells (P5.p and P7.p) divide symmetrically instead of asymmetrically, as both daughters express the LL fate (Figure 11 c). Mutants defective in the /in-l 7or /in-78 genes variably disrupt the asymmetry of one of these ECT cell divisions (by P7.p). In some individuals this division is symmetric, generating two daughters that express the same abnormal (ABN) fate, which is different from both of the fates normally expressed by the two daughters of this division (Figure 11 c). In other /in-l 7 or /in-78 individuals this ECT cell division is reversed in its polarity, so that the anterior daughter expresses the fate normally expressed by the posterior daughter, and vice versa. Since the h-26, h-7 7, h-1 7, and lin-78 mutations characterized all reduce or eliminate gene activity (Ferguson and Horvitz, 1985), the functions of these four genes are normally needed to make certain sister cells different from each other. The effects of h-77 have been characterized further (Sternberg and Horvitz, 1988). Mutations in /in-77 not only affect the vulva1 cell lineages but also cause at least five other types of ectodermal and gonadal blast cells that normally divide asymmetrically to divide symmetrically instead. In addition, /in-77 mutations cause sister cells to be equal in size as well as equivalent in developmental fate. These observations suggest that the M-7 7 product acts in mother cells as a component of a mechanism that establishes the asymmetry of specific asymmetric cell divisions. The asymmetric cell divisions affected by /in-l 7, /in-77,
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(a) Two sequential asymmetric cell divisions occur during the postembryonic development of the C. elegans vulva (Sulston and Horvitz, 1977; Sternberg and Hot-&z, 1986). First, each of three P ectodermal blast cells P5-P7 divides to generate an anterior neuroblast (Pna, called NB in this figure) and a posterior ectoblast (Pn.p, called ECT). Two of these ectoblasts, P5.p and P7.p, then divide to generate two different types of ectoblasts, the anterior of which is known as LL and the posterior of which is known as TN. (b) In the fin-26 mutant, the sister cells generated by the P cells both become neuroblasts (Ferguson et al., 1987). (c) In /in-l7 mutants, the sister cells generated by P5.p and P7.p both become LL ceUs (Ferguson et al., 1987). In M-77 and /in-78 mutants, the sister cells generated by P7.p often have identical fates that are different from both of the normal fates of the daughters of this division. (d) During embryonic development of the C. elegans nervous system, an asymmetric cell division generates the PHB sensory neuron and the HSN motor neuron (Sulston et al., 1983). By contrast, a ham-f mutant seems to generate two identical sister cells that have characteristics of both the PHB and HSN neuron types (Desai et al., ISEIS).
and /in-18 seem likely to be intrinsically determined, since the fates of the daughter cells produced have not been altered by the killing of other cells. For example, the two sister cells (LL and TN) generated by the divisions of the P5.p and P7.p cells do not seem to constitute an equivalence group, since the killing of one does not cause the other to express its fate (Sternberg and Horvitz, 1988). However, the failure of such experiments to reveal cryptic developmental potentials necessarily constitutes negative evidence, and it remains conceivable that cell interactions function in specifying these cell fates. The variable abnormalities seen in /in-17 and /in-18 mutants could be explained if mutations in these genes perturb cytoplasmic localization within the mother cell so that either the polarity of the distribution of specific developmental determinants is reversed or such determinants become mixed causing
both daughter cells to express the same, but abnormal, fate. The gene lin-ll encodes a putative transcription factor, which contains a homeodomain and two copies of a cysteine-rich motif called LIM (Freyd et al., 1990). It seems likely that the Lin-1 1 protein acts to cause differential gene expression between the sister cells generated by the divisions of the P5.p and P7.p cells, and that these differences in gene expression result in the different fates of these sister cells. What might cause /in-l1 to be active in only one of the two sister cells LL and TN? One possibility emerges from the observation that the LIM domain of the Lin-11 protein binds two atoms of zinc and four atoms each of iron and free sulfur in a redox-sensitive iron-sulfur cluster (Li et al., 1991). Thus, Lin-I 1 activity might be regulated by changes in redox potential that occur during the course of the vulva1 cell lineages. In principle, h-11 might act in the mother cell to produce RNAs or proteins that are differentially distributed to the two daughter cells, might be expressed in the mother cell with either its own RNA or protein products being asymmetrically distributed to one of the two daughter cells, or might be expressed in only one of the two daughter cells. Since /in-l 1 affects the same cell division in vulva1 development as does/in-l 7, the/in-l 7 (and also the /in-18) gene might act in the mother cell with In-17 or act in one of the daughter cells in response to /in- 17. The C. elegans gene ham-l (HSN abnormal migration) also affects asymmetric cell division (Desai et al., 1988). ham-l was discovered in a screen for mutants abnormal in the two serotonergic HSN motor neurons, which are required for egg laying. In ham-l animals, the presumptive HSN neurons do not undergo their normal migrations apparently because they fail to acquire a proper HSN identity. ham-l mutants often have two extra HSN neurons, Interestingly, ham-l mutants also often lack the sisters of the HSN neurons, the PHB sensory neurons, which suggests that the PHB neurons are transformed to express the fate of their sister cells. However, in ham-l animals the HSN neurons are abnormal, presumably because they are partially transformed into PHB neurons. Thus, the two daughters of each PHB-HSN mother cell seem to express characteristics of both the PHB neuron and the HSN neuron (Figure lld). One plausible interpretation of the Ham-l phenotype is that the asymmetric division of the PHB-HSN mother cell is intrinsically determined and the distribution of developmental determinants from the mother cell to its daughters is perturbed in ham-l animals. If so, ham-l functions in the localization or segregation of developmental determinants. Alternatively, if the PHB-HSN division is regulated by cell interactions, ham-l could function in a cell signaling process responsible for determining the fates of sister cells. Two other C. elegans genes that function in asymmetric cell division warrant mention. Mutations in these genes change an asymmetric cell division of one type into an asymmetric cell division of another type. The /in-44 gene, identified on the basis of its effects on the development of the male tail, specifies the polarity of certain asymmetric cell divisions, i.e., in /in-44 animals sister cells express
Review: 251
Mechanisms
of Asymmetric
(a) wild
Cell Division
type
(b) numb
mutants
0
-+ 60 S
(c)
S
oversensitive
mutants
0 d GN
Figure
12. Drosophila
Figure based on Uemura et al. (1989) and Jan and Jan (1990). (a) In the embryonic development of the peripheral nervous system of Drosophila, the precursor cell that generates an es organ appears to divide asymmetrically to form one structural cell precursor(S) and one precursor that generates a glial cell and a neuron (GN). (b) In the numb mutant, the es precursor cell generates two structural cell precursors. (c) In the oversensitive mutant, the es precursor cell generates two glial-neural precursors.
each other’s normal fates (Horvitz, 1990; Herman, 1991). Thus, if an anterior daughter normally is of type A and the posterior daughter of type B, in a /in-44 mutant the anterior daughter can be of type B and the posterior daughter of type A. In-44 could act either before cell division to define the polarity of the mother cell or after cell division to define the fates of the daughter cells. The unc86(uncoordinated) gene, which was discovered because it affects a number of behaviors, causes one of the two sister cells generated by the asymmetric division of certain neuroblasts to become different from its mother cell (Horvitz and Sulston, 1980; Chalfie et al., 1981). Thus, if a mother cell of type C normally divides to make daughters of types A and B, in an uric-86 mutant that cell can divide to make daughters of types A and C. The Uric-86 protein is a member of the POU family of transcription factors and is expressed specifically within the daughter cell it affects, suggesting that it controls gene expression in that cell to make it different from its mother (Finney et al., 1988; Finney and Ruvkun, 1990).
Fruit Fly Studies of the development of the Drosophila nervous system have led to the identification of two genes that appear to function in asymmetric cell division. Mutants defective in the numb gene lack most of the neurons of the embryonic peripheral nervous system and have more than the normal number of support cells usually associated with these neurons (Uemura et al., 1989). For example, a simple external sensory or “es” organ, which is thought to be chemosensory or mechanosensory in function, normally consists of one neuron, one glial cell, and two support cells. These four cells are probably generated by two rounds of cell division by a single precursor cell. In numb animals, four cells are present in each es organ, but all four are support cells. Since the two support cells seem to be derived from the sister of the cell that generates the glial cell and neuron, numb mutations probably transform the glial-neuroblast to express the fate of its sister cell, the support cell precursor (Figure 12). It is not known whether the asymmetric division of the es organ precursor cell is intrinsically determined or whether the two daughter cells it produces become different as a consequence of cell interactions. Interestingly, mutations in the oversensitive gene have effects that are opposite to those of mutations in thenumb gene (Jan and Jan, 1990). Specifically, loss of function of the oversensitive gene causes the support cell precursor to express the fate of the glial-neuroblast precursor (Figure 12). Thus, each of these sister cell fates requires the function of a specific gene for its expression, so that neither fate can be regarded as a simple default state. Presumably, the numb and oversensitive genes act in a negative regulatory pathway that specifies the fates of these sister cells, as do the SW/ and SIN genes in budding yeast. The order of action of these Drosophila genes within such a pathway should be revealed by further genetic analyses. Conclusions Although the fundamental basis of asymmetric cell division is not yet understood at a mechanistic level for any of the examples described above, enough is known to establish that a variety of distinct mechanisms are used to make sister cells different from each other. In some cases sister cells are different from the time they are generated, whereas in other cases initially identical sister cells become different as a consequence of cell interactions. The intrinsic differences between sister cells can be determined either by differences in their cytoplasmic factors (B. subtilis, budding yeast, and the nematode AB and PI cells) or by differences in their chromosomes (fission yeast, possibly Caulobacter and Anabaena). It should be noted that these mechanisms need not be mutually exclusive, and it remains possible that both cytoplasmic and chromosomal differences function in certain asymmetric cell divisions. Cell signaling seems to be able to affect the asymmetry of cell division either via direct cell-cell contact (nematode ABa and ABp cells) or via a diffusible molecule (Volvox). Cell signaling can even act to cause intrinsically asymmetric cell divisions to be symmetric instead (Anabaena, pos-
Cdl 252
sibly Volvox). In addition, cell signaling can play a role in inducing further differentiation of sister ceils that are intrinsically determined to be different (6. subtilis, budding yeast, possibly fission yeast). The examples we have discussed reveal that many classes of molecules function to make sister cells different from each other. Transcription factors, and other DNAbinding proteins such as components of chromatin, act in 1 of 2 sister cells to regulate differentially the expression of batteries of genes responsible for particular differentiated characteristics (B. subtilis, budding yeast, nematode). Such transcription factors are themselves regulated by other cell type-specific transcription factors (B. subtilis). Nucleases catalyze genomic rearrangements and proteases activate proproteins in processes necessary for the differentiation of 1 of 2 sister cell types (B. subtilis, budding yeast, fission yeast, possibly Anabaena). Receptors and other components of signal transduction systems allow cells to respond appropriately to their sisters or their neighbors (budding yeast, possibly fission yeast, and nematode ABa and ABp cells). Proteins in the cytoskeletal apparatus are responsible for determining the location and plane of cell division as well as the distribution of cytoplasmic determinants (nematode AB and PI cells, possibly Volvox). Other proteins function in marking DNA strands (fission yeast). Extension of the studies described above, as well as others not considered here because of space and thematic constraints, should provide further insights into the biochemical functions of these and other proteins that act in asymmetric cell divisions. More generally, the findings from each experimental system should help in the analysis of many of the others. The mechanisms that control asymmetric cell division in B. subtilis and in budding yeast have been presented in some detail because they exemplify the fact that none of the genes involved is acting in isolation; rather, each functions within an extensive regulatory cascade. Thus, as asymmetric cell divisions are further analyzed in multicellular organisms, it must be anticipated that the genes responsible will prove to encode components of pathways or possibly even of intricate networks of interacting genes. Indeed, multiple genes affecting single asymmetric cell divisions have already been identified in both nematodes (/in-17, /in-77, /in-78) and flies (numb, oversensitive). To understand how any gene functions in the control of asymmetric cell division, it will be necessary to elucidate both how it acts and how it is regulated. Genes that act in such pathways might be distinguished on the basis of whether they function in differentiation or in the generation of asymmetry per se. Consider again the cell division depicted in Figure 1, in which a cell divides to make sisters of different types A and B. If a mutation that results in a loss of gene function causes that division to generate two cells of type B, the gene defined by that mutation must be necessary for the sister cells to express different fates. Such a gene could act either to cause the asymmetry of the division or to effect that asymmetry once it has been specified. A gene required for expression of the differentiated B fate-whether or not a cell expressing that fate was generated by an asymmetric cell division-
would be of the latter class. myoD provides a plausible example of such a differentiation factor (Weintraub et al., 1989). Many of the genes discussed above could function similarly. By contrast, the C. elegans gene lin-77 affects the fates of a variety of distinct cell types and in each case acts to make sister cells different from each other. Thus, /in-l 7 seems likely to control the specification of the asymmetry of these cell divisions. Only by comprehensive analyses of both single genes and complete pathways of genes will both the determinants and the executors of asymmetric cell division be revealed. Acknowledgments We are deeply indebted to Erik Jorgensen for preparing the diagrams at the last moment. We thank Alan Grossman, Bob Haselkorn, Yuh Nung Jan, Ken Kemphues, David Kirk, Amar Klar, Rich Losick, Austin Newton, Lucy Shapiro, and membersof the Horvitz laboratory for many helpful suggestions concerning this manuscript. H. Fl. H. is an Investigator of the Howard Hughes Medical Institute.
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