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Mechanisms of asymmetric cell division during animal development
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Mechanisms of asymmetric cell division during animal development Juergen A Knoblich Recent years have seen the discovery of machineries for asymmetric cell division in a number of different organisms. The Inscuteable protein is a central component of such a machinery in Drosophila. Within dividing Drosophila neural precursor cells, Inscuteable directs both the orientation of the mitotic spindle and the asymmetric segregation of the proteins Numb, Prospero and Miranda into one of the two daughter cells. Numb can act by repressing signalling via the transmembrane receptor Notch, whereas Miranda localizes the transcription factor Prospero which initiates daughter cell specific gene expression. The identification of Numb homologs in other species has suggested that this machinery might be conserved from Drosophila to vertebrates.
Addresses Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria; e-mail: [email protected] Current Opinion in Cell Biology 1997, 9:833-841 http://biomednet.com/elecref/0955067400900833
© Current Biology Ltd ISSN 0955-0674 Abbreviations aa amino acids CNS centralnervous system ES externalsensory GMC ganglion mother cell HAM-1 HSN abnormal migration-1 PNS peripheralnervous system PTB phosphotyrosine-binding SOP sensory organ precursor Su(H) Suppressor of Hairless
Introduction
All multicellular organisms start as single cells. During development, the progeny of this cell differentiate into a large variety of different cell types. Two distinct mechanisms are employed to generate this diversity (for a review, see [1]). A number of identical cells may be generated initially and these cells may assume different fates later by interacting with each other, interacting with neighboring cells or responding to diffusible factors released by neighboring cells. Alternatively, intrinsically asymmetric cell divisions can create daughter cells that are different from their time of birth. Such asymmetric divisions (I will use the term 'asymmetric cell divisions' instead of 'intrinsically asymmetric cell divisions' in this review) can be generated when a polarized mother cell is capable of segregating determinants into one of its two daughter cells to initiate a particular developmental pathway in this cell, but not in its sister cell.
Even though the concept of asymmetric cell division was formulated more than a hundred years ago (see references in [1]), we have only recently begun to understand asymmetric cell division at a molecular level (see Table 1 for a summary of the most important model systems that have contributed to this recent progress). Molecular mechanisms of asymmetric cell division during animal development have been analyzed mainly in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. During C. elegans development, the division of the zygote is asymmetric and the genes PAR-l--6 and NMY-2 [2"] are involved in generating this asymmetry. This process has recently been extensively reviewed [3] and I want to focus here on asymmetric cell divisions that occur later during development. T h e Drosophila nervous system has proven to be an excellent model system with which to study asymmetric cell division during tissue formation. T h e genes numb, prospero, inscuteable and miranda are part of one machinery for asymmetric cell division and will be discussed below. I will also discuss the recent discovery of numb homologs in vertebrates [4"'] and in C. elegans (G Garriga, personal communication), which has suggested that a similar machinery exists in other animals. Finally, I will describe the ham-1 gene, which might be part of a related mechanism that functions during C. elegans nervous system development.
Asymmetric cell division during Drosophila development Numb and Prospero: some background
T h e development of both the Drosophila peripheral nervous system (PNS) and the Drosophila central nervous system (CNS) involves a number of asymmetric cell divisions. T h e proteins N u m b and Prospero have been shown to play important roles during these divisions (Table 1). T h e numb gene was identified by study of a mutation that affects the development of external sensory (ES) organs and other sensory structures of the Drosophila PNS [5]. ES organs consist of two outer cells (hair and socket cells) and two inner cells (neuron and sheath cells) which arise during development from a single sensory organ precursor (SOP) cell (Figure la). In wild-type embryos, the SOP cell divides into a IIA cell, which is the precursor of the two outer cells, and a liB cell, which is the precursor of the two inner cells [6]. In a numb mutant, however, the SOP cell divides into two IIA cells, which give rise to four outer cells and no inner cells [5]. Conversely, when the numb gene is overexpressed in SOP cells they divide into two liB cells [7]. numb encodes a membrane-associated protein that contains an amino-terminal phosphotyrosine-binding (PTB) domain.
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Cell differentiation
Table 1 Some Important model systems for study of asymmetric cell dMslo. In bacteria, yeast and animals. Organism/tissue
Daughter cell fates
Mechanism of asymmetry
Bacillus subtilis
During starvation, cells divide into a larger mother cell and a smeller forespore. Later, the mother cell completely engulfs the forespore and ultimately degenerates after the foreapore has differentiated into a mature spore,
Lack of nutrients triggers a histidine kinese cascade that ultimately phoephorylatas the master regulator Spo0A, which induces division and turns on mother-cell- and foreapore-specific transcription factors o"E and oF. Preferential activation of the SpollE phoaphatase on the foreapore side of the septum leads to activation of oF in the forespore and later of o E in the mother cell.
References" [47]t
Caulobacter crescentus
Cell division creates a motile swarmer cell with a rotary flagellum and a non-motile stalked cell.
Cell cycle progression activates the master regulator CtrA and induces division. The kinase RbE is localized at the division plane and is selectively captured by the swarmer cell compartment during septation, leading to selective phosphorylation and activation of the transcription factor RbD in this compartment.
[47]t
S. cerevisiae
The larger daughter cell (which is usually called the 'mother cell') transcribes the HO endonucleaso and is capable of mating-type switching. The smaller daughter cell does not transcribe the HO gane and cannot switch its mating type.
The ASH1 protein, which is only present in the daughter cell nucleus, functions as a transcriptional represser of the HO gene. Ashl RNA is transported into the daughter cell by a actin/myosin-dependent machinery including the proteins SHE1-5.
[23]t[25°']
C. e/egans zygote and germline
The first cell division during C. elegans development produces a larger anterior AB cell and a smaller posterior P1 cell, which differ in developmental potential, cell cycle timing, protein expression and spindle orientation. The P granules segregate specifically into the P1 cell.
Generation of asymmetry requires the genes par1-6 end mex-l. The PAR-f and PAR-2 proteins localize to the posterior cortex of the zygote before division and segregate into the P1 cell. PAR-3 localizes to the anterior cortex and segregates into the AB cell. Localization of PAR1-3 requires the nonmuscle myosin NMY-2.
[2"114"]t
C. elegans nervous system (HSN and PHB netlrons)
The HSN/PHB neuroblast divides into an anterior cell which undergoes apoptosis and a posterior HSN/PHB precursor. The HSN/PHB precursor later divides into the HSN and PHB neurons.
Correct lineage specification in the HSN/PHB lineage requires the ham-1 gone. In ham-1 mutants, both daughters of the HSN/PHB neuroblast frequently survive and produce extra neurons. The division of the HSN/PHB precursor is also affected. HAM-1 localizes asymmetrically to the posterior codex, suggesting that it does not itself act as a segregating determinant, but is required for the segregation of such a determinant to the opposite, anterior side of the cell.
[44,45"]
Drosophila nervous system
PNS: the SOP divides into a ltA cell (precursor of hair and sucket cell) and a liB cell (precursor of neuron and sheath cell). CNS: the neuroblast divides into another neuroblast and the GMC, which in turn divides into two neurons.
The proteins Numb, Prospero and Miranda segregate into the GMC (CNS) or into the liB cell (PNS). In neuroblasts, this process requires inscuteab/e. Numb represses Notch signalling, Miranda transports Prospero and Prospero functions as a transcription factor.
[18",20"1142,481 t
Drosophila oogenesis (stem cell divisions)
Germline stem cells divide asymmetrically into another stem cell and a cytoblast, which gives rise to nurse cells and the oooyte. The spectrosome, a spectrin-rich spherical cytoplasmic structure, segregates asymmetrically into the stem cell and is required for spindle orientation.
Stem cell divisions are regulated by signals from surrounding somatic ceils, presumably via the hedgehog or wingless pathway. Maintenance of stem cell fate does not require an intact spectrosome, but does require the genes pumilio and piwi.
[49,501
Vertebrate cortex ventricular zone
Ceils in the ventricular zone divide perpendicularly to the surface into an apical daughter cell that remains in the ventricular zone and a basal daughter cell that migrates towards the cortical plate and presumably differentiates into a neuron. In contrast, divisions parallel to the surface are symmetric and both daughter cells remain in the ventricular zone.
The mouse Numb protein is asymmetrically localized to the apical side during both symmetric and asymmetric divisions. Cell division perpendicular to the surface results in unequal distribution of Numb, whereas division parallel to the surface results in equal distribution. A function for Numb during these asymmetric divisions remains to be shown.
[421t
"3his column includes recent reviews (marked by t) and important recent publications.
In dividing SOP cells, N u m b localizes asymmetrically into a cortical crescent in the membrane area overlying one of the two spindle poles. As a result, it is preferentially segregated into one of the two daughter cells in telophase [7,8]. T h e s e results suggest that the higher concentration of N u m b protein in one SOP daughter cell leads to the establishment of a liB cell fate in this cell and makes it different from its sister cell.
A similar function for N u m b has more recently been demonstrated during Drosophila C N S development [9] (Figure lb). In a wild-type embryo, the MP2 neuroblast divides into a dorsal/basal dMP2 neuron and a ventral/apical vMP2 neuron which can be distinguished from each other both by their gene expression pattern and by the projection pattern of their axons. In a numb mutant, MP2 forms two vMP2 cells, whereas numb overexpression
Mechanismsof asymmetricceil divisionduringanimal developmentKnoblich 835
Figure 1
Cell lineage
mutant
numb
numb
overexpression
(a) (i)
(ii)
C
, ~ Socket
SOP 0
(iii)
T ~,~B Hair
(iv)
SOP
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@
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liB
Sheath Neuron
Outer cells
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IIA
Inner cells
IIA
(b) (i)
dMP2 neuron
!
(ii)
(iii)
MP2
vM% 4¢M,
(iv)
MP2
o vM P2
o vM P2
MP2
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@ dMP2
vMP2 neuron
Current Opinion in Cell Biology
Asymmetric cell divisions in the Drosophila nervous system that involve numb function. (a) (i) The Drosophila ES organ. (ii) The four ceils that form a Drosophila ES organ arise during development in a series of asymmetric cell divisions. The SOP cell divides into a IIA cell and a lib cell. Later, the IIA cell gives rise to the two outer cells, the hair and the socket cells, whereas the liB cell gives rise to the two inner cells, the neuron and the sheath cell. (iii) In a numb mutant, the SOP cell divides into two IIA cells, whereas (iv) after overexpression of numb the SOP cell divides into two liB cells. (b) (I) The Drosophila dorsal (d)MP2 neuron, which projects an axon posteriorly and expresses orthodenticle, and ventral (v)MP2 neuron, which projects an axon anteriorly and does not express orthodenticle, are shown. (ii) The Drosophila MP2 neuroblast divides into the dMP2 neuron and the vMP2 neuron. (iii) In a numb mutant, MP2 produces two vMP2 neurons, and (iv) after numb overexpression MP2 produces two dMP2 neurons.
leads to the generation of two dMP2 cells. T h e N u m b protein segregates into the dMP2 daughter cell during division, suggesting that its higher concentration in the dorsal daughter cell initiates the dMP2 cell fate. Most Drosophila neuroblasts, however, show a cell division pattern that is considerably different from that of MP2. T h e y usually divide along the apical-basal axis into a largerapical daughter cell and a basal ganglion mother cell (GMC). T h e G M C divides once more into two postmitotic neurons or gila precursors, while the apical daughter cell retains neuroblast characteristics and continues to divide in a stem cell like fashion. T h e N u m b protein preferentially segregates into the basal GMC during mitosis, but in numb mutants GMCs are not transformed
into additional neuroblasts [7]. In contrast, the prospero gene has been shown to play an important role in the specification of certain GMCs. In prospero mutants, some GMCs fail to express GMC-specific genes (such as even-skipped [10]) and continue to express genes that are usually neuroblast-specific (such as deadpan [11]). However, they are not completely transformed into their sister cells: they still divide only once more and produce two neurons. Prospero encodes a transcription factor with limited homology to homeodomain proteins [10-12]. Similarly to Numb, Prospero segregates asymmetrically in dividing Drosophila neural precursor cells. In neuroblasts, the Prospero protein can first be detected shortly before mitosis, when it starts to accumulate at the apical side of the cell [13] (see Figure 2). During mitosis, Prospero
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Cell differentiation
Figure 2
Mitosis (metaphase)
Interphase
Apical
~
Basal
Inspcutteiable
@--~Neurobiast
(~
~
~
After mitosis
~
4, Epidermis Neuroblast GMC
(~
Tight apical crescent
Tightapical crescent
Not detectable (degraded or delocalized)
Membrane-localized, homogeneously distributed
Tight basal crescent (requiresinscuteable)
Cortex o basal daughter cell (GMC)
Starts appearing weakly on apical cortex
Tight basal crescent (requiresinscuteable)
Membrane of basal daughter cell (GMC), then rapidly degraded
protein
Prospero protein
(~
~
Mem~rar~i~ ( ~
Starts appearing weakly on apical cortex
Tight basal crescent (requiresinscuteable and miranda)
ne, then nucleus, of basal daughter cell (GMC)
Strong apical localization (cortex or cytoplasm)
Basal crescent (requiresinscuteable and staufen)
Cortex of basal daughter cell (GMC)
prospero
Current Opinion in Cell Biology
Asymmetric localization of proteins and RNAs in Drosophila neuroblasts during asymmetric cell division. Drosophila neuroblasts arise from epithelial cells and segregate basally from the epithelial cell layer (top left). After segregation, they divide asymmetrically along the apical-basal axis (top center) into a larger apical cell that retains neuroblast characteristics and a smaller basal ganglion mother cell (GMC) (top right). This figure summarizes recent results on the asymmetric localization of the proteins Inscuteable, Numb, Miranda and Prospero, and of the prospero RNA. Black areas in the bottom section of the figure denote localization of these proteins and RNA.
colocalizes with Numb into a crescent at the basal cell cortex, and, together with Numb, it segregates into the GMC [8,13,14]. After mitosis, Prospero translocates into the GMC nucleus, whereas Numb is retained at the cell membrane. Prospero localization and Numb localization are independent of each other, but their strikingly similar localization suggests that both proteins are transported by a common localization machinery. Some components of this localization machinery have been identified during the past year and I will discuss them below.
Asymmetric localization of Prospero: RNA localization or a localized adaptor?
Proteins can localize asymmetrically when the corresponding RNA transcripts are transported to one side of a cell where they are translated locally (for a review on RNA localization, see [15]). During Drosophila oogenesis, for example, a complex RNA localization machinery is used to localize bicoicl and nanos RNA to the anterior and posterior poles of the oocyte, respectively (for a review on Drosophila oogenesis, see [16]). T h e RNA-binding protein
Mechanisms of asymmetric cell division during animal development Knoblich
Staufen is one of the major components of this machinery [17]. Recent experiments have revealed some intriguing parallels in RNA localization during Drosophila oogenesis and during asymmetric division in Drosophila neuroblasts (Figure 2). The prospero RNA is asymmetrically localized to the apical cell cortex in interphase neuroblasts and relocalizes to the basal cell cortex during mitosis ([18°]; A Schuldt, C Davidson, A Brand, personal communication; CQ Doe, personal communication). Staufen is expressed not only during Drosophila oogenesis, but also in dividing Drosophila neuroblasts [171. The Staufen protein is found at the neuroblast cortex with a higher concentration at the apical side during interphase [18"] and at the basal side during mitosis ([19]; A Schuldt, C Davidson, A Brand, personal communication). In staufen mutants, the prospero RNA is frequently delocalized throughout the cytoplasm and fails to translocate to the basal cell cortex ([18°]; A Schuldt, C Davidson, A Brand, personal communication; CQ Doe, personal communication). However, the Prospero protein is still correctly localized and staufen mutants have no detectable nervous system defects and develop into viable adult flies. Even though the absence of nervous system defects could be explained by perdurance of maternal Staufen protein, it is more likely that prospero RNA localization is not required for normal development under laboratory conditions, though it might increase the efficiency of asymmetric Prospero segregation. T h e recent discovery of the Miranda protein has demonstrated that asymmetric localization of Prospero occurs at the protein level [20°°]. Miranda was identified in a two-hybrid screen for proteins that bind to the Prospero localization domain [14} and it encodes a coiled-coil protein of approximately 800 amino acids (aa) [20°°]. Miranda is expressed in both neuroblasts and SOP cells during asymmetric cell division. During mitosis, the protein co-localizes with Prospero into a cortical crescent (Figure 2). After mitosis, it enters the same daughter cell as Prospero and is rapidly degraded when Prospero translocates into the nucleus. In miranda-deficient embryos, Prospero is never localized to the cell membrane and is homogeneously distributed throughout the cytoplasm instead. Miranda-deficient embryos have no defects in Numb localization, even though the Miranda protein can also bind Numb in vitro. Thus, Miranda functions as a membrane anchor for Prospero that mediates its asymmetric localization, possibly by linking Prospero to the same machinery that localizes Numb. In contrast to the manner of Prospero localization in
Drosophila, recent experiments in Saccharomyces cerevisiae have shown that segregation of a determinant during asymmetric cell division can be achieved by RNA localization. During mitosis in S. cerevisiae, the transcriptional repressor Ashlp is produced in only one of the two daughter cells, where it represses transcription of the 110 locus and thus inhibits the ability to switch the mating type ([21°°,22°']; for a recent review, see [23]). The process of asymmetric
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Ashlp accumulation requires a transport machinery that includes the type V myosin Myo4p/Shelp [24°°]. A series of experiments has recently provided convincing evidence that the ASH1 mRNA is transported by this machinery and that RNA localization is the mechanism that generates Ashlp protein asymmetry [25°°,26]. Mutations that are known to disrupt ASH1 RNA localization also disrupt the localization of the Ashlp protein and the ASH1 RNA can be visualized in the process of tracking through the bud into the daughter cell, while the protein accumulates in the daughter cell nucleus [25°°].
Inscuteable directs localization of Numb and Prospero and spindle orientation
The asymmetric localization of Numb, Prospero and Miranda is both temporally and spatially correlated with the orientation of the mitotic spindle ([8]; CP Shen, personal communication): both processes occur during prophase of mitosis, and the Numb, Prospero and Miranda crescents always form over one of the two spindle poles. However, asymmetric protein localization and spindle orientation are independent events: neither numb nor prospero mutants have defects in spindle orientation, and when the mitotic spindle is disrupted by microtubule drugs Numb and Prospero are still localized [8]. It thus appears that several independent events during asymmetric cell division are directed by a common axis of asymmetry [8], a concept that has previously been postulated for the earliest cell divisions during C. elegans development [27]. The Inscuteable protein plays a key role in establishing this axis of asymmetry in Drosophila [28",29]. Inscuteable is a 859aa protein that contains a putative Src homology 3 (SH3)-binding domain [29]. Like Numb, Prospero and Miranda, the Inscuteable protein is asymmetrically localized in dividing neuroblasts and SOP cells. However, Inscuteable localization precedes Numb, Prospero and Miranda localization and occurs to the opposite (in neuroblasts apical) side of the cell (Figure 2). In contrast to Numb, Prospero and Miranda, which become localized during prophase of mitosis [8,13,14,20°°], Inscuteable is already localized into an apical cortical crescent during late interphase [28°°]. Inscuteable localization does not require Numb, Prospero or Miranda. However, in the absence of Inscuteable, neuroblasts, which normally divide along the apical-basal axis, divide with a random division plane, and the Numb, Prospero and Miranda crescents form at random positions around the circumference of the cell [20°',28°']. Numb, Prospero and Miranda still co-localize, indicating that their localization machineries have common elements downstream of inscuteable. Conversely, when inscuteable is expressed in ectodermal epithelial cells which normally divide parallel to the surface, those cells reorient their mitotic spindle and divide perpendicularly to the epithelial surface [28°°]. However, they do not localize Numb asymmetrically, indicating that other elements of
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Cell differentiation
the Numb
localization
machinery
are absent
from these
CdlS.
A careful analysis of the insnrteablc mutant phenotype show5 that SOP cells, in contrast to neuroblasts, have no defects in spindle orientation and Numb/Prospero localization, even though Inscuteable is expressed and asymmetrically localized in these cells [ZS*.,ZS]. In contrast to nemoblasts, SOP cells divide parallel to the epithelial surface, suggesting that inscu~eable is only required for asymmetric cell divisions that occur along the apical-basal axis. The observation that Inscuteable specifically localizes to the apical side when ectopically expressed in epithelial cells in the prospective epidermis [28**], the developing hindgut and the salivary gland (]A Knoblich, unpublished data) further links Inscuteable localization to epithelial polarity. Neuroblasts arise during development from polarized epithelial cells and Inscuteable might recognize inherited epithelial polarity to orient their division along the apical-basal axis. However, more direct experiments are required to support this hypothesis. The function of Inscuteable is not restricted to mitotic cells. The Staufen protein, which localizes apically in wild-type interphase neuroblasts, becomes delocalized in inscuie&e mutants, leading co a defect in the basal transport of the prospero RNA during mitosis [18*]. The fact that inscuteubf’ is expressed in nonmitotic cells during oogenesis and in the developing wing veins [ZS] further suggests that Inscureable functions are not restricted to mitosis. One might speculate that Inscuteable induces the polarization of some cytoskeletal structure during interphase and that this polarity is recognized during mitosis by the machinery for spindle orientation and Numb/Prosper0 localization. Downstream of Numb and Prospero: how different protein concentrations translate into different cell fates
Even though a direct transcriptional target for the Prosper0 protein remains to be identified, its nuclear localization and homology to a homeodomain-containing protein suggest that Prospero acts by directly activating or repressing expression of particular gene5 in one of the two daughter cells. Candidate targets for Prosper0 are the genes pueir-skipped and deadpan, whose expression pattern is altered inprospero mutants [lO,ll]. In contrast, the events that occur downstream of Numb are less obvious. In both the SOP and the MPZ lineages, the transmembrane receptor Notch has been shown to function antagonistically to Numb (for a recent review on Notch signalling, see [30]): inactivation of Notch transforms the HA cell into an additional IIB cell or the vMP2 neuron into an additional dMPZ n+ron [31,32’]. #OR/~ genetically acts downstream of numb (32’,33*], and it has recently become clear that Numb functions by inactivating Notch signalling in one of the two daughter cells [32*-34.1, a mechanism that has been suggested before [35]. Normally, the receptor Notch is activated by
one of its ligands, Delta or Serrate, which are present on the cell surface of neighboring cells. During bristle development, Delta is likely to act as a ligand for Notch [36], but the issue of a redundant function of Serrate remains to be addressed. The Notch signal is transmitted into the nucleus, where it leads to transcriptional activation of the genes of the E~lra~tcer ofSplit complex, presumably by direct binding of the transcription factor Suppressor of Hairless @u(H)) to upstream regions of these genes [37]. Tissue culture experiments have suggested that Notch activity in fact regulates nuclear localization of Su(H) [38]: when A’~bc~and Su(iY} are co-transfected into Drosophda tissue culture cells, Su(H) is retained in the cytoplasm, but when these cells contact neighboring cells that express the Notch ligand Delta, Su(H) can translocate into the nucleus and presumably become transcriptionally active. Even though it is not clear how much this reflects the in v&o situation [39], this tissue culture system can be used as an assay for Notch activity. When the same cells are triple-transfected with Ar&, Su(ff) and numb, Su(H) is retained in the cytoplasm even when Delta-expressing cells are contacted [34*]. This effect requires the PTB domain of the Numb protein which is also essential for Numb function in viva [34-l. In oitro, the PTB domain of Numb can directly bind to the intracellular domain of the Notch receptor [33.]. These experiments suggest that, after localization into the IIB cell {or the dMP2 cell), Numb inhibits Notch signalling in this cell by binding to the intracellular domain of the Notch receptor.
Asymmetric cell division during vertebrate development The recent discovery of a mouse numb homolog [4.-j has suggested that mechanisms similar to the ones described above might function during vertebrate nervous system development. Asymmetric cell divisions during vertebrate cortical development occur in the ventricular zone of the developing cortex {Table 1). Video timelapse experiments in ferret cortical slice cultures have shown that asymmetric cell divisions preferentially occur with a mitotic spindle oriented perpendicularly to the ventricular surface ([40]; for reviews, see [41,42]). The apical daughter cell remains in the ventricular zone and continues to divide, whereas the basal daughter cell migrates towards the cortical plate, where it differentiates into a neuron. In contrast, cell divisions that are oriented parallel to the surface are mostly symmetric, and both daughter cells remain in the ventricular zone. Mouse Numb is asymmetrically localized in dividing cells of the ventricular zone (4**]. When expressed in Drosophila embryos, the protein co-localizes with Drosophilu Numb into a crescent and is able to rescue the numb mutant phenotype. However, there are some characteristic differences: localization of mouse Numb in the ventricular zone is not correlated with spindle orientation. Mouse Numb localizes to the apical side
Mechanisms of asymmetric cell division during animal development Knoblich
irrespective of the position of the spindle poles [4°°]. This opens an interesting possibility for the generation of asymmetric cell divisions: in cells that divide parallel to the ventricular surface cytokinesis bisects the N u m b crescent, resulting in equal amounts of N u m b protein in the two daughter cells, whereas a division perpendicular to the surface segregates the N u m b crescent into one daughter cell, presumably resulting in an asymmetric cell division. It is intriguing to speculate that a mammalian inscuteable homolog might trigger these differences in spindle orientation. However, Drosophila inscuteable is involved in coordinating spindle orientation and Numb localization, and this coordination is not conserved in vertebrates. A putative mammalian inscuteable homolog would therefore have to interact differently with the mouse N u m b localization machinery. A second sequence homolog of Drosophila N u m b has recently been identified [43°]. Even though this numblike gene is also able to partially rescue the Drosophila numb phenotype, its localization in the cytoplasm, lack of asymmetric localization, and expression in the cortical plate and not in the ventricular zone suggest a function in neuronal differentiation and not in asymmetric cell division. However, numb and numblike share a common downstream target with Drosophila numb: all three proteins can bind to the intracellular domain of the Notch receptor. Thus, both certain aspects of the machinery for asymmetric localization and the downstream signalling events may be conserved between Drosophila numb and its vertebrate counterpart.
HAM-1 directs asymmetric cell divisions during C. elegans nervous system development T h e nematode C. elegans develops in a stereotyped cell lineage that involves many asymmetric cell divisions and therefore presents an ideal model system for studying asymmetric cell divisions. Regulation of the early cell divisions during nematode development (see Table 1) has been reviewed recently [3] and will not be discussed here. During C. elegans nervous system development, the protein HAM-1 (HSN abnormal migration-l) has been shown to play a role in asymmetric cell division (Table 1). In wild-type embryos, the HSN/PHB neuroblast divides into an anterior daughter cell, which undergoes apoptosis, and a posterior HSN/PHB precursor cell, which later gives rise to the neurons HSN and PHB. In ham-1 mutants, the anterior daughter frequently fails to undergo apoptosis and develops into an additional HSN/PHB precursor, producing excess HSN and PHB neurons [44,45°]. HAM-1 encodes a 414 aa protein that is localized to the cell cortex. During division of the HSN/PHB neuroblast, HAM-1 localizes into a crescent at the posterior cortex, suggesting that it is inherited by the posterior daughter cell [45°]. Thus, absence of HAM-1 results in defects in the daughter cell that does not normally inherit the protein. This could be due to interaction between the two daughter
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cells. Alternatively, HAM-1 might not be a segregating determinant itself, but instead might be involved in the segregation of another unknown determinant into the anterior daughter cell. This possibility suggests a functional similarity between Inscuteable in Drosophila and HAM-1 in C. elegans, even though no defects in spindle orientation have been observed in ham-1 mutant embryos. T h e recent discovery of a numb homolog in C. elegans (G Garriga, personal communication) opens up experimental strategies to test such a functional similarity: if HAM-1 is a functional homolog of Inscuteable, the asymmetric localization of C. elegans N u m b should be affected in HAM-1 mutants.
Future directions Exciting progress has been made during the past few years towards understanding how animal cells can divide asymmetrically into two different daughter cells. T h e identification of the Drosophila N u m b protein has initiated the characterization of a mechanism that might be conserved between C. elegans, Drosophila and vertebrates. Despite our progress, many open questions remain. T h e mechanical basis for N u m b (and Miranda) localization is not clear. Actin-myosin-based transport is involved in asymmetrically localizing determinants in S. cerevisiae [24 °-] and C. elegans [2•]. A similar transport machinery could exist in Drosophila, but other mechanisms are possible (discussed in [46]). How does Inscuteable fit into the picture? How does Inscuteable interact with the localization machinery and what is the positional information that directs Inscuteable localization? T h e coming years will certainly see some answers to these questions that might lead to the characterization of a universal mechanism for asymmetric cell division during tissue formation in animal development.
Acknowledgements I want to thank Yuh Nung Jan and Lily Jan for their continuous and generous support. 1 also want to thank the members of the Jan Lab for fruitful discussions and Salim Abdelilah, Claudia Petritsch, Yan Sun, and Weimin Zhong for their comments on the manuscript. I am currently supported by the Howard Hughes Medical Institute.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest =o of outstanding interest 1.
Horvitz HR, Herskowitz h Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question. Ceil 1992, 68:237-255.
2. •
Guo S, Kemphues KJ: A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature 1996, 382:455-458. The NMY-2 gene is identified by screening an expression library with the conserved domain of the PAR-1 protein. Silencing of NMY-2 by RNA injection leads to mislocaJization of PAR-l, PAR-2 and PAR-3, suggesting that NMY-2 is involved in establishing cellular polarity in the C. e/egans zygote. 3.
Guo S, Kemphues KJ: Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo. Curt Opin Genet Dev 1996, 6:408-415.
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4. ••
Zhong W, Feder JN, Jiang MM, Jan LY, Jan YN: Asymmetric localization of a mammalian Numb homolog during mouse cortical neurogenesis. Neuron 1996, 17:43-53. Low stringency hybridization identifies a mammalian homolog of Drosophila numb. Mammalian numb is widely expressed and asymmetrically localized in dividing cells of the ventricular zone in the developing cortex. Mouse Numb localizes asymmetrically when expressed in Drosophila and can rescue the numb mutant phenotype.
a myosin and restricts yeast mating-type switching to mother cells. Cell 1996, 84:599-709. ASHI is identified in a screen for mutations that allow mating-type switching in yeast daughter cells. ASH1 encodes a member of the GATA family of zinc finger transcription factors (see also [22"]). Ashlp functions as a transcriptional repressor of the HO gene, which is required for mating-type switching. During anaphase, Ashlp accumulates to much higher levels in daughter cell nuclei and inhibits mating-type switching in these cells. 22. •.
Sil A, Herskowitz h Identification of asymmetrically localized determinant, Ashlp, required for lineage-specific transcription of the yeast HO gene. Cell 1996, 84:711-722. In this paper, ASH1 is identified in a direct visual screen for mutations which produce daughter ceils that are able to switch their mating type (see also [21"°]). Ashlp is localized to the daughter ceil nucleus, where it inhibits transcription of HO and inhibits mating-type switching. ASH1 overexpression disrupts this asymmetry and inhibits mating-type switching in both the mother and the daughter cell.
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UemuraT, Shepherd S, Ackerman L, Jan LY, Jan YN: numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos, Cell 1989, 58:349-360.
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Spana EP, Doe CCI: The Prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila. Development 1995, 121:3187-3195.
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Kraut R, Chia W, Jan LY, Jan YN, Knoblich JA: Role of inscutaable in orienting asymmetric cell divisions in Drosophila. Nature 1996, 383:50-55. In Drosophila neuroblasts, the Inscuteable protein localizes asymmetrically during interphase, opposite to the side where the Numb and Prospero crescents will form during mitosis. In the absence of Inscuteable, Numb and Prospero are not correctly localized and the mitotic spindle is not oriented correctly. Ectopic expression of inscuteable induces reorientation of the mitotic spindle, but is not sufficient for Numb localization.
RNAs. Cell 1995, 81:161-170.
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Gr~JnertS, St Johnston D: RNA localization and the development of asymmetry during Drosophila oogenesis. Curt Opin Genet Dev 1996, 6:395-402.
17.
St Johnston D, Beuchle D, Nesslein-Volhard C: steufen, a gene required to localize maternal RNAs in the Drosophila egg. Cell 1991, 66:51-63.
18. •
Li P, Yang X, Wasser M, Cal Y, Chia W: Inscuteable and Staufen mediate asymmetric Iocalisation and segregation of Prospero RNA during Drosophila neuroblast cell divisions. Cell 1997, 90:437-447. prospero RNA is asymmetrically localized in dividing Drosophila neuroblasts. Staufen, which is picked up in a two-hybrid screen with Drosophila inscuteable, binds the 3' end of the prospero RNA. Both staufen and inscuteable are required for correct asymmetric localization of the prospero RNA, but only inscuteab/e is required for Prospero protein localization [28"], suggesting that prospero RNA localization is not essential for localization of the Prospero protein. 19.
Broadus JB, Doe CG: Extrinsic cues, intrinsic cues, and microfilaments regulate asymmetric localization of Prospero, Staufen, and Inscuteable in Drosophila neuroblasts. Curt Bio/ 1997, in press.
20. ••
Shen CP, Jan LY, Jan YN: Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila. Cell 1997, 90:449-458. The miranda gene is isolated in a two-hybrid screen by using the Prospero localization domain, miranda is expressed in cells that localize Prospero asymmetrically, and the Miranda protein co-localizes with Prospero throughout most of mitosis. In the absence of miranda, Prospero becomes cytoplasmic, suggesting that Miranda functions as an adaptor that transiently localizes Prospero to the cell membrane during mitosis. 21. ••
Bobola N, Jansen RP, Shin TH, Nasmyth K: Asymmetric accumulation of Ashlp in postanaphase nuclei depends on
23.
Chang F, Drubin DG: Cell division: why daughters cannot be like their mothers. Curt Bio11996, 6:651-654.
24. •.
JansenRP, Dowzer C, Michaelis C, Galore M, Nasmyth K: Mother cell-specific HO expression in budding yeast depends on the unconventional myosin Myo4p and other cytoplasmic proteins. Cell 1996, 84:687-697. SHE1-SHE5 are identified in a screen for genes that are required for expression of the HO endonuclsase in mother cells. In she mutants, mothers fail to switch their mating type. SHE1/MY04 encodes a class V minimyosin; SHE5 has been previously identified as BNI1 and encodes a protein that contains an FH1/2 domain; SHE3 encodes a coiled-coil protein; and SHE2 and SHE4 have no homology to other genes. 25. ,*
Long RM, Singer RH, Meng X, Gonzalez I, Nasmyth K, Jansen RP: Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 1997, 277:383-387. Fluorescent in situ hybridization reveals that the ASH1 RNA is asymmetrically localized in dividing yeast cells. ASH1 RNA localization requires actin, tropomyosin, profi/in and the SHE genes, but not tubulin. The 3' untranslated region of the ASH1 gene is sufficient for its asymmetric localization when fused to/acZ RNA.
28. •.
29.
KrautR, Campos-Ortega JA: inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeleton adaptor protein. Dev Bio11996, 174:65-81.
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Artavanis-Tsakonas S, Matsuno K, Fortini ME: Notch signaling. Science 1995, 268:225-232.
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Hartenstein V, Posakony JW: A dual function of the Notch gene in Drosophila sensUlum development. Dev Biol 1990, 142:1330.
32. Spana EP, Doe CCh Numb antagonizes Notch signaling to • specify sibling neuron call fates. Neuron 1996, 17:21-26. During division of the MP2 neuroblast, absence of Delta or Notch leads to the opposite cell fate transformation to that caused by absence of Numb. Delta and Notch genetically act downstream of Numb, suggesting that Numb acts by inhibiting Notch in one of the two MP2 daughter cells. 33. •
Guo M, Jan LY, Jan YN: Control of daughter cell fates during asymmetric division: interaction of Numb and Notch. Neuron 1996, 17:27-41. During external sensory organ development, reduction of Notch function and reduction of numb function lead to opposite cell fate transformations. Similarly, overexpression of the two genes has opposite effects. Notch genetically acts downstream of numb, but upstream of tramtrack. The Numb phosphotyrosine-binding domain interacts with the Notch intracellular domain in in vitro co-immunoprecipitations and in the yeast two-hybrid assay, suggesting a mechanism for the inhibition of Notch by Numb. 34. •
FriseE, Knoblich JA, Younger-Shepherd S, Jan LY, Jan YN: The Drosophila Numb protein inhibits signaling of the Notch
Mechanisms of asymmetric cell division during animal development Knoblich
receptor during cell-cell interaction in sensory organ lineage. Proc Nat/Acad Sci USA 1996, 93:11925-11932. In a tissue culture system that uses the nuclear localization of Suppressor of Hairless as an assay for Notch activity, Numb can suppress Notch activation by the ligand Delta. Both the Numb phosphotyrosine-binding (PTB) domain and the very carboxyl terminus of the Numb protein are required for this activity. The PTB domain is required for functions downstream of Numb, but not for asymmetric localization of Numb in vivo, suggesting that the in vivo function of numb is to inhibit Notch signaling.
841
suggests distinct roles during mouse cortical neurogenesis. Development 1997, 124:1887-1897. Low stringency hybridization identifies numb/ike, a second vertebrate sequence homolog of Drosophila numb (see also Zhong et a/. I4°']). numblike can partially rescue the numb mutant phenotype when expressed in Drosophila, but Numblike is a cytoplasmic protein that is not asymmetrically segregated during mitosis. Like Numb, it can bind to the Notch1 intracellular domain, suggesting a common downstream function, but its expression pattern in the cortical plate suggests a function during neuronal differentiation rather than asymmetric cell division.
35.
Posakony JW: Nature versus nurture: asymmetric cell divisions in Drosophila bristle developmenL Cell 1994, 76:415-418.
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ParksAL, Muskavitch MA: Delta function is required for bristle organ determination and morphogenesis in Drosophila. Day Biol 1993, 157:484-496.
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Lecourtois M, Schweisguth F: The neurogenic Suppressor of Hairless DNA-binding protein mediates the transcriptional activation of the Enhancer of Split complex genes triggered by Notch signaling. Genes Day 1995, 9:2598-2608.
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FortiniME, Artavanis-Tsakonas S: The Suppressor of Hairless protein participates in Notch receptor signaling. Cell 1994, 79:273-282.
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Gho M, Lecourtois M, Geraud G, Posakony JW, Schweisguth F: Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signaling. Development 1996, 122:1673-1682.
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Knoblich JA, Jan LY, Jan YN: Asymmetric segregation of the Drosophila Numb protein during mitosis: facts and speculations. Cold Spring Harb Symp Guant Biol 1997, in press.
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Chenn A, McConnell SK: Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 1995, 82:631-641.
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Shapiro L, Losick R: Protein localization and cell fate in bacteria. Science 199?, 276:712-718.
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RhyuMS, Knoblich JA: Spindle orientation and asymmetric cell fate. Cell 1995, 82:523-526~
Lin H, Schagat T: Neuroblasts: a model for the asymmetric division of stem cells. Trends Genet 1997, 13:33-39.
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Huttner WB, Brand M: Asymmetric division and polarity of neuroepithelial cells. Curt Opin Neurobiol 1997, 7:29-39.
Lin H, Spradling AC: Fusome asymmetry and oocyte determination in Drosophila. Dev Genet 1995, 16:6-12.
50.
43. •
Zhong W, Jiang MM, Weinmaster G, Jan LY, Jan YN: Differential expression of mammalian numb, numblika and Notch1
Lin H, Spradling AC: A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 1997, 124:2463-2476.
44.
Desai C, Garriga G, Mclntire SL, Horvitz HR: A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature 1988, 336:638-646.
45.
Guenther C, Garriga G: Asymmetric distribution of the C e l e g a n s HAM-1 protein in neuroblasts enables daughter cells to adopt distinct fates. Development 1996, 122:3509-3518. This paper describes the asymmetric localization of the HAM-1 protein during mitosis in the C. elegans HSN/PHB neuroblast. The localization suggests that HAM-1 segregates into the posterior daughter cell, but the anterior daughter cell is more strongly affected in ham.1 mutants. This can be explained if the two daughter cells interact with each other, or if HAM*I is involved in the segregation of another cell fate determinant. •
Mechanisms of asymmetric cell division during animal development Juergen A Knoblich Recent years have seen the discovery of machineries for asymmetric cell division in a number of different organisms. The Inscuteable protein is a central component of such a machinery in Drosophila. Within dividing Drosophila neural precursor cells, Inscuteable directs both the orientation of the mitotic spindle and the asymmetric segregation of the proteins Numb, Prospero and Miranda into one of the two daughter cells. Numb can act by repressing signalling via the transmembrane receptor Notch, whereas Miranda localizes the transcription factor Prospero which initiates daughter cell specific gene expression. The identification of Numb homologs in other species has suggested that this machinery might be conserved from Drosophila to vertebrates.
Addresses Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria; e-mail: [email protected] Current Opinion in Cell Biology 1997, 9:833-841 http://biomednet.com/elecref/0955067400900833
© Current Biology Ltd ISSN 0955-0674 Abbreviations aa amino acids CNS centralnervous system ES externalsensory GMC ganglion mother cell HAM-1 HSN abnormal migration-1 PNS peripheralnervous system PTB phosphotyrosine-binding SOP sensory organ precursor Su(H) Suppressor of Hairless
Introduction
All multicellular organisms start as single cells. During development, the progeny of this cell differentiate into a large variety of different cell types. Two distinct mechanisms are employed to generate this diversity (for a review, see [1]). A number of identical cells may be generated initially and these cells may assume different fates later by interacting with each other, interacting with neighboring cells or responding to diffusible factors released by neighboring cells. Alternatively, intrinsically asymmetric cell divisions can create daughter cells that are different from their time of birth. Such asymmetric divisions (I will use the term 'asymmetric cell divisions' instead of 'intrinsically asymmetric cell divisions' in this review) can be generated when a polarized mother cell is capable of segregating determinants into one of its two daughter cells to initiate a particular developmental pathway in this cell, but not in its sister cell.
Even though the concept of asymmetric cell division was formulated more than a hundred years ago (see references in [1]), we have only recently begun to understand asymmetric cell division at a molecular level (see Table 1 for a summary of the most important model systems that have contributed to this recent progress). Molecular mechanisms of asymmetric cell division during animal development have been analyzed mainly in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. During C. elegans development, the division of the zygote is asymmetric and the genes PAR-l--6 and NMY-2 [2"] are involved in generating this asymmetry. This process has recently been extensively reviewed [3] and I want to focus here on asymmetric cell divisions that occur later during development. T h e Drosophila nervous system has proven to be an excellent model system with which to study asymmetric cell division during tissue formation. T h e genes numb, prospero, inscuteable and miranda are part of one machinery for asymmetric cell division and will be discussed below. I will also discuss the recent discovery of numb homologs in vertebrates [4"'] and in C. elegans (G Garriga, personal communication), which has suggested that a similar machinery exists in other animals. Finally, I will describe the ham-1 gene, which might be part of a related mechanism that functions during C. elegans nervous system development.
Asymmetric cell division during Drosophila development Numb and Prospero: some background
T h e development of both the Drosophila peripheral nervous system (PNS) and the Drosophila central nervous system (CNS) involves a number of asymmetric cell divisions. T h e proteins N u m b and Prospero have been shown to play important roles during these divisions (Table 1). T h e numb gene was identified by study of a mutation that affects the development of external sensory (ES) organs and other sensory structures of the Drosophila PNS [5]. ES organs consist of two outer cells (hair and socket cells) and two inner cells (neuron and sheath cells) which arise during development from a single sensory organ precursor (SOP) cell (Figure la). In wild-type embryos, the SOP cell divides into a IIA cell, which is the precursor of the two outer cells, and a liB cell, which is the precursor of the two inner cells [6]. In a numb mutant, however, the SOP cell divides into two IIA cells, which give rise to four outer cells and no inner cells [5]. Conversely, when the numb gene is overexpressed in SOP cells they divide into two liB cells [7]. numb encodes a membrane-associated protein that contains an amino-terminal phosphotyrosine-binding (PTB) domain.
834
Cell differentiation
Table 1 Some Important model systems for study of asymmetric cell dMslo. In bacteria, yeast and animals. Organism/tissue
Daughter cell fates
Mechanism of asymmetry
Bacillus subtilis
During starvation, cells divide into a larger mother cell and a smeller forespore. Later, the mother cell completely engulfs the forespore and ultimately degenerates after the foreapore has differentiated into a mature spore,
Lack of nutrients triggers a histidine kinese cascade that ultimately phoephorylatas the master regulator Spo0A, which induces division and turns on mother-cell- and foreapore-specific transcription factors o"E and oF. Preferential activation of the SpollE phoaphatase on the foreapore side of the septum leads to activation of oF in the forespore and later of o E in the mother cell.
References" [47]t
Caulobacter crescentus
Cell division creates a motile swarmer cell with a rotary flagellum and a non-motile stalked cell.
Cell cycle progression activates the master regulator CtrA and induces division. The kinase RbE is localized at the division plane and is selectively captured by the swarmer cell compartment during septation, leading to selective phosphorylation and activation of the transcription factor RbD in this compartment.
[47]t
S. cerevisiae
The larger daughter cell (which is usually called the 'mother cell') transcribes the HO endonucleaso and is capable of mating-type switching. The smaller daughter cell does not transcribe the HO gane and cannot switch its mating type.
The ASH1 protein, which is only present in the daughter cell nucleus, functions as a transcriptional represser of the HO gene. Ashl RNA is transported into the daughter cell by a actin/myosin-dependent machinery including the proteins SHE1-5.
[23]t[25°']
C. e/egans zygote and germline
The first cell division during C. elegans development produces a larger anterior AB cell and a smaller posterior P1 cell, which differ in developmental potential, cell cycle timing, protein expression and spindle orientation. The P granules segregate specifically into the P1 cell.
Generation of asymmetry requires the genes par1-6 end mex-l. The PAR-f and PAR-2 proteins localize to the posterior cortex of the zygote before division and segregate into the P1 cell. PAR-3 localizes to the anterior cortex and segregates into the AB cell. Localization of PAR1-3 requires the nonmuscle myosin NMY-2.
[2"114"]t
C. elegans nervous system (HSN and PHB netlrons)
The HSN/PHB neuroblast divides into an anterior cell which undergoes apoptosis and a posterior HSN/PHB precursor. The HSN/PHB precursor later divides into the HSN and PHB neurons.
Correct lineage specification in the HSN/PHB lineage requires the ham-1 gone. In ham-1 mutants, both daughters of the HSN/PHB neuroblast frequently survive and produce extra neurons. The division of the HSN/PHB precursor is also affected. HAM-1 localizes asymmetrically to the posterior codex, suggesting that it does not itself act as a segregating determinant, but is required for the segregation of such a determinant to the opposite, anterior side of the cell.
[44,45"]
Drosophila nervous system
PNS: the SOP divides into a ltA cell (precursor of hair and sucket cell) and a liB cell (precursor of neuron and sheath cell). CNS: the neuroblast divides into another neuroblast and the GMC, which in turn divides into two neurons.
The proteins Numb, Prospero and Miranda segregate into the GMC (CNS) or into the liB cell (PNS). In neuroblasts, this process requires inscuteab/e. Numb represses Notch signalling, Miranda transports Prospero and Prospero functions as a transcription factor.
[18",20"1142,481 t
Drosophila oogenesis (stem cell divisions)
Germline stem cells divide asymmetrically into another stem cell and a cytoblast, which gives rise to nurse cells and the oooyte. The spectrosome, a spectrin-rich spherical cytoplasmic structure, segregates asymmetrically into the stem cell and is required for spindle orientation.
Stem cell divisions are regulated by signals from surrounding somatic ceils, presumably via the hedgehog or wingless pathway. Maintenance of stem cell fate does not require an intact spectrosome, but does require the genes pumilio and piwi.
[49,501
Vertebrate cortex ventricular zone
Ceils in the ventricular zone divide perpendicularly to the surface into an apical daughter cell that remains in the ventricular zone and a basal daughter cell that migrates towards the cortical plate and presumably differentiates into a neuron. In contrast, divisions parallel to the surface are symmetric and both daughter cells remain in the ventricular zone.
The mouse Numb protein is asymmetrically localized to the apical side during both symmetric and asymmetric divisions. Cell division perpendicular to the surface results in unequal distribution of Numb, whereas division parallel to the surface results in equal distribution. A function for Numb during these asymmetric divisions remains to be shown.
[421t
"3his column includes recent reviews (marked by t) and important recent publications.
In dividing SOP cells, N u m b localizes asymmetrically into a cortical crescent in the membrane area overlying one of the two spindle poles. As a result, it is preferentially segregated into one of the two daughter cells in telophase [7,8]. T h e s e results suggest that the higher concentration of N u m b protein in one SOP daughter cell leads to the establishment of a liB cell fate in this cell and makes it different from its sister cell.
A similar function for N u m b has more recently been demonstrated during Drosophila C N S development [9] (Figure lb). In a wild-type embryo, the MP2 neuroblast divides into a dorsal/basal dMP2 neuron and a ventral/apical vMP2 neuron which can be distinguished from each other both by their gene expression pattern and by the projection pattern of their axons. In a numb mutant, MP2 forms two vMP2 cells, whereas numb overexpression
Mechanismsof asymmetricceil divisionduringanimal developmentKnoblich 835
Figure 1
Cell lineage
mutant
numb
numb
overexpression
(a) (i)
(ii)
C
, ~ Socket
SOP 0
(iii)
T ~,~B Hair
(iv)
SOP
/ ~ ' ~ O
@
@
lib
liB
Sheath Neuron
Outer cells
SOP
IIA
Inner cells
IIA
(b) (i)
dMP2 neuron
!
(ii)
(iii)
MP2
vM% 4¢M,
(iv)
MP2
o vM P2
o vM P2
MP2
@ dMP2
@ dMP2
vMP2 neuron
Current Opinion in Cell Biology
Asymmetric cell divisions in the Drosophila nervous system that involve numb function. (a) (i) The Drosophila ES organ. (ii) The four ceils that form a Drosophila ES organ arise during development in a series of asymmetric cell divisions. The SOP cell divides into a IIA cell and a lib cell. Later, the IIA cell gives rise to the two outer cells, the hair and the socket cells, whereas the liB cell gives rise to the two inner cells, the neuron and the sheath cell. (iii) In a numb mutant, the SOP cell divides into two IIA cells, whereas (iv) after overexpression of numb the SOP cell divides into two liB cells. (b) (I) The Drosophila dorsal (d)MP2 neuron, which projects an axon posteriorly and expresses orthodenticle, and ventral (v)MP2 neuron, which projects an axon anteriorly and does not express orthodenticle, are shown. (ii) The Drosophila MP2 neuroblast divides into the dMP2 neuron and the vMP2 neuron. (iii) In a numb mutant, MP2 produces two vMP2 neurons, and (iv) after numb overexpression MP2 produces two dMP2 neurons.
leads to the generation of two dMP2 cells. T h e N u m b protein segregates into the dMP2 daughter cell during division, suggesting that its higher concentration in the dorsal daughter cell initiates the dMP2 cell fate. Most Drosophila neuroblasts, however, show a cell division pattern that is considerably different from that of MP2. T h e y usually divide along the apical-basal axis into a largerapical daughter cell and a basal ganglion mother cell (GMC). T h e G M C divides once more into two postmitotic neurons or gila precursors, while the apical daughter cell retains neuroblast characteristics and continues to divide in a stem cell like fashion. T h e N u m b protein preferentially segregates into the basal GMC during mitosis, but in numb mutants GMCs are not transformed
into additional neuroblasts [7]. In contrast, the prospero gene has been shown to play an important role in the specification of certain GMCs. In prospero mutants, some GMCs fail to express GMC-specific genes (such as even-skipped [10]) and continue to express genes that are usually neuroblast-specific (such as deadpan [11]). However, they are not completely transformed into their sister cells: they still divide only once more and produce two neurons. Prospero encodes a transcription factor with limited homology to homeodomain proteins [10-12]. Similarly to Numb, Prospero segregates asymmetrically in dividing Drosophila neural precursor cells. In neuroblasts, the Prospero protein can first be detected shortly before mitosis, when it starts to accumulate at the apical side of the cell [13] (see Figure 2). During mitosis, Prospero
836
Cell differentiation
Figure 2
Mitosis (metaphase)
Interphase
Apical
~
Basal
Inspcutteiable
@--~Neurobiast
(~
~
~
After mitosis
~
4, Epidermis Neuroblast GMC
(~
Tight apical crescent
Tightapical crescent
Not detectable (degraded or delocalized)
Membrane-localized, homogeneously distributed
Tight basal crescent (requiresinscuteable)
Cortex o basal daughter cell (GMC)
Starts appearing weakly on apical cortex
Tight basal crescent (requiresinscuteable)
Membrane of basal daughter cell (GMC), then rapidly degraded
protein
Prospero protein
(~
~
Mem~rar~i~ ( ~
Starts appearing weakly on apical cortex
Tight basal crescent (requiresinscuteable and miranda)
ne, then nucleus, of basal daughter cell (GMC)
Strong apical localization (cortex or cytoplasm)
Basal crescent (requiresinscuteable and staufen)
Cortex of basal daughter cell (GMC)
prospero
Current Opinion in Cell Biology
Asymmetric localization of proteins and RNAs in Drosophila neuroblasts during asymmetric cell division. Drosophila neuroblasts arise from epithelial cells and segregate basally from the epithelial cell layer (top left). After segregation, they divide asymmetrically along the apical-basal axis (top center) into a larger apical cell that retains neuroblast characteristics and a smaller basal ganglion mother cell (GMC) (top right). This figure summarizes recent results on the asymmetric localization of the proteins Inscuteable, Numb, Miranda and Prospero, and of the prospero RNA. Black areas in the bottom section of the figure denote localization of these proteins and RNA.
colocalizes with Numb into a crescent at the basal cell cortex, and, together with Numb, it segregates into the GMC [8,13,14]. After mitosis, Prospero translocates into the GMC nucleus, whereas Numb is retained at the cell membrane. Prospero localization and Numb localization are independent of each other, but their strikingly similar localization suggests that both proteins are transported by a common localization machinery. Some components of this localization machinery have been identified during the past year and I will discuss them below.
Asymmetric localization of Prospero: RNA localization or a localized adaptor?
Proteins can localize asymmetrically when the corresponding RNA transcripts are transported to one side of a cell where they are translated locally (for a review on RNA localization, see [15]). During Drosophila oogenesis, for example, a complex RNA localization machinery is used to localize bicoicl and nanos RNA to the anterior and posterior poles of the oocyte, respectively (for a review on Drosophila oogenesis, see [16]). T h e RNA-binding protein
Mechanisms of asymmetric cell division during animal development Knoblich
Staufen is one of the major components of this machinery [17]. Recent experiments have revealed some intriguing parallels in RNA localization during Drosophila oogenesis and during asymmetric division in Drosophila neuroblasts (Figure 2). The prospero RNA is asymmetrically localized to the apical cell cortex in interphase neuroblasts and relocalizes to the basal cell cortex during mitosis ([18°]; A Schuldt, C Davidson, A Brand, personal communication; CQ Doe, personal communication). Staufen is expressed not only during Drosophila oogenesis, but also in dividing Drosophila neuroblasts [171. The Staufen protein is found at the neuroblast cortex with a higher concentration at the apical side during interphase [18"] and at the basal side during mitosis ([19]; A Schuldt, C Davidson, A Brand, personal communication). In staufen mutants, the prospero RNA is frequently delocalized throughout the cytoplasm and fails to translocate to the basal cell cortex ([18°]; A Schuldt, C Davidson, A Brand, personal communication; CQ Doe, personal communication). However, the Prospero protein is still correctly localized and staufen mutants have no detectable nervous system defects and develop into viable adult flies. Even though the absence of nervous system defects could be explained by perdurance of maternal Staufen protein, it is more likely that prospero RNA localization is not required for normal development under laboratory conditions, though it might increase the efficiency of asymmetric Prospero segregation. T h e recent discovery of the Miranda protein has demonstrated that asymmetric localization of Prospero occurs at the protein level [20°°]. Miranda was identified in a two-hybrid screen for proteins that bind to the Prospero localization domain [14} and it encodes a coiled-coil protein of approximately 800 amino acids (aa) [20°°]. Miranda is expressed in both neuroblasts and SOP cells during asymmetric cell division. During mitosis, the protein co-localizes with Prospero into a cortical crescent (Figure 2). After mitosis, it enters the same daughter cell as Prospero and is rapidly degraded when Prospero translocates into the nucleus. In miranda-deficient embryos, Prospero is never localized to the cell membrane and is homogeneously distributed throughout the cytoplasm instead. Miranda-deficient embryos have no defects in Numb localization, even though the Miranda protein can also bind Numb in vitro. Thus, Miranda functions as a membrane anchor for Prospero that mediates its asymmetric localization, possibly by linking Prospero to the same machinery that localizes Numb. In contrast to the manner of Prospero localization in
Drosophila, recent experiments in Saccharomyces cerevisiae have shown that segregation of a determinant during asymmetric cell division can be achieved by RNA localization. During mitosis in S. cerevisiae, the transcriptional repressor Ashlp is produced in only one of the two daughter cells, where it represses transcription of the 110 locus and thus inhibits the ability to switch the mating type ([21°°,22°']; for a recent review, see [23]). The process of asymmetric
837
Ashlp accumulation requires a transport machinery that includes the type V myosin Myo4p/Shelp [24°°]. A series of experiments has recently provided convincing evidence that the ASH1 mRNA is transported by this machinery and that RNA localization is the mechanism that generates Ashlp protein asymmetry [25°°,26]. Mutations that are known to disrupt ASH1 RNA localization also disrupt the localization of the Ashlp protein and the ASH1 RNA can be visualized in the process of tracking through the bud into the daughter cell, while the protein accumulates in the daughter cell nucleus [25°°].
Inscuteable directs localization of Numb and Prospero and spindle orientation
The asymmetric localization of Numb, Prospero and Miranda is both temporally and spatially correlated with the orientation of the mitotic spindle ([8]; CP Shen, personal communication): both processes occur during prophase of mitosis, and the Numb, Prospero and Miranda crescents always form over one of the two spindle poles. However, asymmetric protein localization and spindle orientation are independent events: neither numb nor prospero mutants have defects in spindle orientation, and when the mitotic spindle is disrupted by microtubule drugs Numb and Prospero are still localized [8]. It thus appears that several independent events during asymmetric cell division are directed by a common axis of asymmetry [8], a concept that has previously been postulated for the earliest cell divisions during C. elegans development [27]. The Inscuteable protein plays a key role in establishing this axis of asymmetry in Drosophila [28",29]. Inscuteable is a 859aa protein that contains a putative Src homology 3 (SH3)-binding domain [29]. Like Numb, Prospero and Miranda, the Inscuteable protein is asymmetrically localized in dividing neuroblasts and SOP cells. However, Inscuteable localization precedes Numb, Prospero and Miranda localization and occurs to the opposite (in neuroblasts apical) side of the cell (Figure 2). In contrast to Numb, Prospero and Miranda, which become localized during prophase of mitosis [8,13,14,20°°], Inscuteable is already localized into an apical cortical crescent during late interphase [28°°]. Inscuteable localization does not require Numb, Prospero or Miranda. However, in the absence of Inscuteable, neuroblasts, which normally divide along the apical-basal axis, divide with a random division plane, and the Numb, Prospero and Miranda crescents form at random positions around the circumference of the cell [20°',28°']. Numb, Prospero and Miranda still co-localize, indicating that their localization machineries have common elements downstream of inscuteable. Conversely, when inscuteable is expressed in ectodermal epithelial cells which normally divide parallel to the surface, those cells reorient their mitotic spindle and divide perpendicularly to the epithelial surface [28°°]. However, they do not localize Numb asymmetrically, indicating that other elements of
838
Cell differentiation
the Numb
localization
machinery
are absent
from these
CdlS.
A careful analysis of the insnrteablc mutant phenotype show5 that SOP cells, in contrast to neuroblasts, have no defects in spindle orientation and Numb/Prospero localization, even though Inscuteable is expressed and asymmetrically localized in these cells [ZS*.,ZS]. In contrast to nemoblasts, SOP cells divide parallel to the epithelial surface, suggesting that inscu~eable is only required for asymmetric cell divisions that occur along the apical-basal axis. The observation that Inscuteable specifically localizes to the apical side when ectopically expressed in epithelial cells in the prospective epidermis [28**], the developing hindgut and the salivary gland (]A Knoblich, unpublished data) further links Inscuteable localization to epithelial polarity. Neuroblasts arise during development from polarized epithelial cells and Inscuteable might recognize inherited epithelial polarity to orient their division along the apical-basal axis. However, more direct experiments are required to support this hypothesis. The function of Inscuteable is not restricted to mitotic cells. The Staufen protein, which localizes apically in wild-type interphase neuroblasts, becomes delocalized in inscuie&e mutants, leading co a defect in the basal transport of the prospero RNA during mitosis [18*]. The fact that inscuteubf’ is expressed in nonmitotic cells during oogenesis and in the developing wing veins [ZS] further suggests that Inscureable functions are not restricted to mitosis. One might speculate that Inscuteable induces the polarization of some cytoskeletal structure during interphase and that this polarity is recognized during mitosis by the machinery for spindle orientation and Numb/Prosper0 localization. Downstream of Numb and Prospero: how different protein concentrations translate into different cell fates
Even though a direct transcriptional target for the Prosper0 protein remains to be identified, its nuclear localization and homology to a homeodomain-containing protein suggest that Prospero acts by directly activating or repressing expression of particular gene5 in one of the two daughter cells. Candidate targets for Prosper0 are the genes pueir-skipped and deadpan, whose expression pattern is altered inprospero mutants [lO,ll]. In contrast, the events that occur downstream of Numb are less obvious. In both the SOP and the MPZ lineages, the transmembrane receptor Notch has been shown to function antagonistically to Numb (for a recent review on Notch signalling, see [30]): inactivation of Notch transforms the HA cell into an additional IIB cell or the vMP2 neuron into an additional dMPZ n+ron [31,32’]. #OR/~ genetically acts downstream of numb (32’,33*], and it has recently become clear that Numb functions by inactivating Notch signalling in one of the two daughter cells [32*-34.1, a mechanism that has been suggested before [35]. Normally, the receptor Notch is activated by
one of its ligands, Delta or Serrate, which are present on the cell surface of neighboring cells. During bristle development, Delta is likely to act as a ligand for Notch [36], but the issue of a redundant function of Serrate remains to be addressed. The Notch signal is transmitted into the nucleus, where it leads to transcriptional activation of the genes of the E~lra~tcer ofSplit complex, presumably by direct binding of the transcription factor Suppressor of Hairless @u(H)) to upstream regions of these genes [37]. Tissue culture experiments have suggested that Notch activity in fact regulates nuclear localization of Su(H) [38]: when A’~bc~and Su(iY} are co-transfected into Drosophda tissue culture cells, Su(H) is retained in the cytoplasm, but when these cells contact neighboring cells that express the Notch ligand Delta, Su(H) can translocate into the nucleus and presumably become transcriptionally active. Even though it is not clear how much this reflects the in v&o situation [39], this tissue culture system can be used as an assay for Notch activity. When the same cells are triple-transfected with Ar&, Su(ff) and numb, Su(H) is retained in the cytoplasm even when Delta-expressing cells are contacted [34*]. This effect requires the PTB domain of the Numb protein which is also essential for Numb function in viva [34-l. In oitro, the PTB domain of Numb can directly bind to the intracellular domain of the Notch receptor [33.]. These experiments suggest that, after localization into the IIB cell {or the dMP2 cell), Numb inhibits Notch signalling in this cell by binding to the intracellular domain of the Notch receptor.
Asymmetric cell division during vertebrate development The recent discovery of a mouse numb homolog [4.-j has suggested that mechanisms similar to the ones described above might function during vertebrate nervous system development. Asymmetric cell divisions during vertebrate cortical development occur in the ventricular zone of the developing cortex {Table 1). Video timelapse experiments in ferret cortical slice cultures have shown that asymmetric cell divisions preferentially occur with a mitotic spindle oriented perpendicularly to the ventricular surface ([40]; for reviews, see [41,42]). The apical daughter cell remains in the ventricular zone and continues to divide, whereas the basal daughter cell migrates towards the cortical plate, where it differentiates into a neuron. In contrast, cell divisions that are oriented parallel to the surface are mostly symmetric, and both daughter cells remain in the ventricular zone. Mouse Numb is asymmetrically localized in dividing cells of the ventricular zone (4**]. When expressed in Drosophila embryos, the protein co-localizes with Drosophilu Numb into a crescent and is able to rescue the numb mutant phenotype. However, there are some characteristic differences: localization of mouse Numb in the ventricular zone is not correlated with spindle orientation. Mouse Numb localizes to the apical side
Mechanisms of asymmetric cell division during animal development Knoblich
irrespective of the position of the spindle poles [4°°]. This opens an interesting possibility for the generation of asymmetric cell divisions: in cells that divide parallel to the ventricular surface cytokinesis bisects the N u m b crescent, resulting in equal amounts of N u m b protein in the two daughter cells, whereas a division perpendicular to the surface segregates the N u m b crescent into one daughter cell, presumably resulting in an asymmetric cell division. It is intriguing to speculate that a mammalian inscuteable homolog might trigger these differences in spindle orientation. However, Drosophila inscuteable is involved in coordinating spindle orientation and Numb localization, and this coordination is not conserved in vertebrates. A putative mammalian inscuteable homolog would therefore have to interact differently with the mouse N u m b localization machinery. A second sequence homolog of Drosophila N u m b has recently been identified [43°]. Even though this numblike gene is also able to partially rescue the Drosophila numb phenotype, its localization in the cytoplasm, lack of asymmetric localization, and expression in the cortical plate and not in the ventricular zone suggest a function in neuronal differentiation and not in asymmetric cell division. However, numb and numblike share a common downstream target with Drosophila numb: all three proteins can bind to the intracellular domain of the Notch receptor. Thus, both certain aspects of the machinery for asymmetric localization and the downstream signalling events may be conserved between Drosophila numb and its vertebrate counterpart.
HAM-1 directs asymmetric cell divisions during C. elegans nervous system development T h e nematode C. elegans develops in a stereotyped cell lineage that involves many asymmetric cell divisions and therefore presents an ideal model system for studying asymmetric cell divisions. Regulation of the early cell divisions during nematode development (see Table 1) has been reviewed recently [3] and will not be discussed here. During C. elegans nervous system development, the protein HAM-1 (HSN abnormal migration-l) has been shown to play a role in asymmetric cell division (Table 1). In wild-type embryos, the HSN/PHB neuroblast divides into an anterior daughter cell, which undergoes apoptosis, and a posterior HSN/PHB precursor cell, which later gives rise to the neurons HSN and PHB. In ham-1 mutants, the anterior daughter frequently fails to undergo apoptosis and develops into an additional HSN/PHB precursor, producing excess HSN and PHB neurons [44,45°]. HAM-1 encodes a 414 aa protein that is localized to the cell cortex. During division of the HSN/PHB neuroblast, HAM-1 localizes into a crescent at the posterior cortex, suggesting that it is inherited by the posterior daughter cell [45°]. Thus, absence of HAM-1 results in defects in the daughter cell that does not normally inherit the protein. This could be due to interaction between the two daughter
839
cells. Alternatively, HAM-1 might not be a segregating determinant itself, but instead might be involved in the segregation of another unknown determinant into the anterior daughter cell. This possibility suggests a functional similarity between Inscuteable in Drosophila and HAM-1 in C. elegans, even though no defects in spindle orientation have been observed in ham-1 mutant embryos. T h e recent discovery of a numb homolog in C. elegans (G Garriga, personal communication) opens up experimental strategies to test such a functional similarity: if HAM-1 is a functional homolog of Inscuteable, the asymmetric localization of C. elegans N u m b should be affected in HAM-1 mutants.
Future directions Exciting progress has been made during the past few years towards understanding how animal cells can divide asymmetrically into two different daughter cells. T h e identification of the Drosophila N u m b protein has initiated the characterization of a mechanism that might be conserved between C. elegans, Drosophila and vertebrates. Despite our progress, many open questions remain. T h e mechanical basis for N u m b (and Miranda) localization is not clear. Actin-myosin-based transport is involved in asymmetrically localizing determinants in S. cerevisiae [24 °-] and C. elegans [2•]. A similar transport machinery could exist in Drosophila, but other mechanisms are possible (discussed in [46]). How does Inscuteable fit into the picture? How does Inscuteable interact with the localization machinery and what is the positional information that directs Inscuteable localization? T h e coming years will certainly see some answers to these questions that might lead to the characterization of a universal mechanism for asymmetric cell division during tissue formation in animal development.
Acknowledgements I want to thank Yuh Nung Jan and Lily Jan for their continuous and generous support. 1 also want to thank the members of the Jan Lab for fruitful discussions and Salim Abdelilah, Claudia Petritsch, Yan Sun, and Weimin Zhong for their comments on the manuscript. I am currently supported by the Howard Hughes Medical Institute.
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