Polarity Cues Research Articles

Asymmetric cell division and axis formation in the embryo

Asymmetric cell divisions play an important role in generating diversity during metazoan development. In the early C. elegans embryo, a series of asymmetric divisions are crucial for establishing the three principal axes of the body plan (AP, DV, LR) and for segregating determinants that specify cell fates. In this review, we focus on events in the one-cell embryo that result in the establishment of the AP axis and the first asymmetric division. We first describe how the sperm-derived centrosome initiates movements of the cortical actomyosin network that result in the polarized distribution of PAR proteins. We then briefly discuss how components acting downstream of the PAR proteins mediate unequal segregation of cell fate determinants to the anterior blastomere AB and the posterior blastomere P1. We also review how a heterotrimeric G protein pathway generates cortically based pulling forces acting on astral microtubules, thus mediating centrosome and spindle positioning in response to AP polarity cues. In addition, we briefly highlight events involved in establishing the DV and LR axes. The DV axis is established at the four-cell stage, following specific cell-cell interactions that occur between P2 and EMS, the two daughters of P1, as well as between P2 and ABp, a daughter of AB. The LR axis is established shortly thereafter by the division pattern of ABa and ABp. We conclude by mentioning how findings made in early C. elegans embryos are relevant to understanding asymmetric cell division and pattern formation across metazoan evolution.

Polarity precedes adhesion

Epithelial cells distinguish top from bottom even before they stick to their neighbors, according to results on page 135 from Harris and Peifer. Figure Apical restriction of DE-Cadherin (green, left) is lost in baz mutants (right). Polarized epithelia are characterized by complexes that provide tight adhesions between neighboring cells. These complexes, known as adherens junctions (AJs), contain transmembrane cadherins that link adjacent cells. Cadherin is anchored to the actin cytoskeleton through the AJ proteins Armadillo and α-catenin. The building of AJs is normally considered the earliest known event in establishing apicobasal polarity. Looking for polarity in the absence of AJs, however, is tricky; sheet architecture falls apart if AJs are removed, so the cells are no longer epithelial. But Harris and Peifer find that, in the developing fly, enough epithelial cells stay in place to determine that a polarized epithelium can develop without AJs. The signs of polarization come from two apical scaffold complexes that also help establish polarity. One apical complex is defined by Bazooka (Baz), which recruits Crumbs, a member of the second complex. The authors see that both complexes attain apical localization in Armadillo mutants. In these flies, an early epithelium is built, and portions of the embryo undergo morphogenesis. Without Baz, on the other hand, AJ components are not correctly localized, suggesting that Baz is the earlier polarity factor. AJs are needed to maintain polarity, as most epithelial structures later broke down in their absence. These defects probably reflect failures in cell–cell adhesion, but AJs also had specific effects on late polarity events, such as the segregation of the basolateral protein Discs Large, which was retained apically the residual epithelial structures of the mutants. The earliest polarity cue is still a mystery, but cells may somehow sense the outside of the embryo and define it as apical. Actin and microtubules were polarized in AJ mutants, suggesting that they may transport Baz to the apical domain. Large complexes of Baz oligomers may then recruit AJ proteins, but as yet it is unclear exactly how Baz is linked to AJ formation.

Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization.

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreased recruitment of endogenous VASP to the leading edge and resulted in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue.

Moving while sticking

Intercalation—the slotting of cells in between one another—is an established method for converting short and fat into long and slim. This is all very well in slippery, adhesion-sparse mesenchymal tissues. But many such extension events occur in epithelial tissues, where cells are glued together by adhesion complexes. Claire Bertet, Lawrence Sulak, and Thomas Lecuit (IBDM, Marseille, France) now report that epithelial intercalation relies on some strategic tugging by myosin that remodels junctions. Figure Contraction and expansion of membranes reorganizes cells as the tissue extends (top to bottom). Anterior is left; posterior is right. The French group expected that extension in fly embryos would involve a few individual motile cells nosing between other stationary cells. Instead they saw “a global and ordered reorganization,” says Lecuit. Borders between cells that lay anterior and posterior to each other contracted to a point. Perpendicular expansion of this point generated a border between dorsal and ventral cells, thus pushing the anterior cell more anterior and the posterior cell more posterior. Extension was therefore achieved by a geometrical shuffling of the original hexagonal arrangement. Myosin II was concentrated near the shrinking (anterior–posterior) membranes and reduced near the expanding (dorsal–ventral) membranes. In embryos with less myosin II, junctions froze and intercalation failed. Myosin need only destabilize adhesion proteins at anterior–posterior membranes, as junction proteins are naturally very dynamic. It is not known how myosin is concentrated preferentially near anterior and posterior membranes. Genes that define anterior–posterior polarity of the fly embryo are needed; they may exert this effect by either local (cell-to-cell) or global (gradient) messages. These polarity cues result in an intercalation method that, says Lecuit, maintains “the balance between stability and dynamics.” It can account for a nearly twofold extension. If more extension is needed, for a gut tube or an arm, then cell shape changes and oriented cell division may come into play. ▪ Reference: Bertet, C., et al. 2004. Nature. 429:667–671. [PubMed]

First cell fate decisions and spatial patterning in the early mouse embryo.

A growing body of evidence indicates that although the early mouse embryo retains flexibility in responding to perturbations, its patterning is initiated at the earliest developmental stages. There are a few spatial cues that are able to influence the pattern of cleavage divisions: one of these lies in the vicinity of the previous meiotic division, the second is associated with the sperm entry and, related to this, the third is the cell shape. Furthermore, the first cleavage separates the zygote into two cells that tend to follow distinguishable fates: one contributes mainly to the embryonic part of the blastocyst, and the other to the abembryonic. The cumulative effect of the early asymmetries generated through cleavage might lead to asymmetric interactions between the first lineages of cells. This could influence development of patterning after implantation. These early polarity cues serve to bias patterning and not as definitive determinants.

Breaking Cellular Symmetry along Planar Axes in Drosophila and Vertebrates

In many organs, epithelial cells are polarized not only along the apicobasal axis, but also along a second axis within a plane. Acquisition of the latter polarity, known as planar cell polarity (PCP) or tissue polarity, is crucial for specialized cellular functions. Genetic programming of PCP has been most thoroughly studied in Drosophila, which has allowed identification of a number of regulatory molecules that are evolutionally conserved. One group of the regulators is responsible for interpreting a hypothetical polarity cue and directing local cytoskeletal reorganization. This group includes a seven-pass transmembrane cadherin known as Flamingo (also known as Starry night), other receptors, and downstream components; and many of those molecules are redistributed to restricted subcellular compartments. Recent studies on a trio of cell-surface molecules challenge a previous hypothesis about the identity of the polarity cue and prompt a novel hypothesis about a global input. Studies on vertebrate systems support the notion that the molecular mechanisms demonstrated in Drosophila are applicable to at least two classes of polarized behaviors of vertebrate cells: sensory hair morphogenesis in the inner ear epithelium, and convergent extension movements during gastrulation.

LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling.

G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. In this report, we show that the asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99. Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that LET-99 and Galpha/GPR-1/2 signaling function in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division.

Exclusion of exogenous phosphatidylinositol-3,4,5-trisphosphate from neutrophil-polarizing pseudopodia: stabilization of the uropod and cell polarity.

Although there is accumulating evidence that the generation and localization of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have important functions in neutrophil polarization and chemotaxis, the mechanism of this linkage has yet to be established. Here, using exogenous fluorescent PtdIns(3,4,5)P(3) introduced into the inner leaflet of the neutrophil plasma membrane by a cationic carrier, we show that: first, PtdIns(3,4,5)P(3) uniformly delivered to the neutrophil plasma membrane is excluded from newly forming pseudopodia; second, PtdIns(3,4,5)P(3) translocates to and is immobilized at the pole opposite a stable polarizing pseudopod; third, asymmetric delivery of PtdIns(3,4,5)P(3) to the neutrophil triggers the generation of polarizing pseudopodia at the opposite pole; and finally, PtdIns(3,4,5)P(3) triggers repetitive Ca(2+) signals, the onset of which precedes morphological polarization. These data suggest that translocation and immobilization of PtdIns(3,4,5)P(3) or a 3,x-phosphorylated metabolite in the uropod functions as an important polarization cue that defines neutrophil polarity and stabilizes the generation of pseudopodia at the opposite pole.

GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in cell guidance and chemotaxis during normal and pathological events. uPAR is GPI-anchored and the mechanism by which it transmits intracellular polarity cues across the plasma membrane during directional sensing has not been elucidated. The constitutively recycling endocytic receptor Endo180 forms a trimolecular complex with uPAR in the presence of uPA, hence its alternate name uPAR-associated protein. Here, we demonstrate that Endo180 is a general promoter of random cell migration and has a more specific function in cell chemotaxis up a uPA gradient. Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac. Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not. From these studies, we conclude that Endo180 is a crucial link between uPA–uPAR and setting of the internal cellular compass.

Pulled Aside

Asymmetric divisions are crucial for generating cell diversity in metazoan organisms and require precise coupling between polarity cues and the positioning of the mitotic spindle. Colombo et al. report the discovery of two GoLoco proteins required for asymmetric spindle positioning in Caenorhabditis elegans embryos. By studying the interactions between these proteins and G proteins of the mitotic apparatus, they demonstrate the requirement for G protein signaling to generate asymmetric pulling forces on the mitotic spindle. These forces arise through the uneven distribution of the GoLoco proteins under the control of the anterior-posterior polarity components PAR-2 and PAR-3. K. Colombo, S. W. Grill, R. J. Kimple, F. S. Willard, D. P. Siderovski, P. Gönczy, Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos. Science 300 , 1957-1961 (2003). [Abstract] [Full Text]

Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos.

Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. We found that the GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, were essential for generation of proper pulling forces. GPR-1/2 interacted with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2.

Differential Activation of the DNA Replication Checkpoint Contributes to Asynchrony of Cell Division in C. elegans Embryos

Background: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated.Results: We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. We demonstrate that preferential activation of this checkpoint in the P1 blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Gα signaling.Conclusion: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos.

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.

Establishing cell polarity in development.

Polarity is a common feature of many different cell types, including the Caenorhabditis elegans zygote, the Drosophila oocyte and mammalian epithelial cells. The initial establishment of cell polarity depends on asymmetric cues that lead to reorganization of the cytoskeleton and polarized localization of several cortical proteins that act downstream of the polarization cues. The past year revealed that homologs of the C. elegans par (partitioning defective) genes are also essential for establishing polarity in Drosophila and vertebrate cells. There is growing evidence that the proteins encoded by these genes interact with key regulators of both the actin and the microtubule cytoskeletons.

Bazooka is required for localization of determinants and controlling proliferation in the sensory organ precursor cell lineage in Drosophila.

Asymmetric divisions with two different division orientations follow different polarity cues for the asymmetric segregation of determinants in the sensory organ precursor (SOP) lineage. The first asymmetric division depends on frizzled function and has the mitotic spindle of the pI cell in the epithelium oriented along the anterior-posterior axis, giving rise to pIIa and pIIb, which divide in different orientations. Only the pIIb division resembles neuroblast division in daughter-size asymmetry, spindle orientation along the apical-basal axis, basal Numb localization, and requirement for inscuteable function. Because the PDZ domain protein Bazooka is required for spindle orientation and basal localization of Numb in neuroblasts, we wondered whether Bazooka plays a similar role in the pIIb in the SOP lineage. Surprisingly, Bazooka controls asymmetric localization of the Numb-anchoring protein Pon, but not spindle orientation, in pI and all subsequent divisions. Bazooka also regulates cell proliferation in the SOP lineage; loss of bazooka function results in supernumerary cell divisions and apoptotic cell death.

A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast

The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

Pod-2, along with pod-1, Defines a New Class of Genes Required for Polarity in the Early Caenorhabditis elegans Embryo

The asymmetric division of the one-cell Caenorhabditis elegans zygote gives rise to two cells of different size and fate, thereby establishing the animal's anterior–posterior (a-p) axis. Through genetics, a number of genes required for this polarity have been characterized, but many components remain unidentified. Recently, our laboratory discovered a mutation in the pod-1 gene (for polarity and osmotic defective) that uniquely perturbed polarity and osmotic protection. Here, we describe a new C. elegans polarity gene identified during screens for conditional embryonic lethals. Embryos in which this gene has been mutated show a loss of physical and developmental asymmetries in the one-cell embryo, including the mislocalization of PAR and POD-1 proteins required for early polarity. Furthermore, mutant embryos are osmotically sensitive, allowing us to designate this gene pod-2. Thus, pod-2, along with pod-1, defines a new class of C. elegans polarity genes. Genetic analyses indicate that pod-2 functions in the same pathway as pod-1. Temperature-shift studies indicate that pod-2 is required during oogenesis, indicating that aspects of embryonic polarization may precede fertilization. pod-2 mutant embryos also exhibit a unique germline inheritance defect in which germline identity localizes to the wrong spot in the one-cell embryo and is therefore inherited by the wrong cell at the four-cell stage. Our data suggest that pod-2 may be required to properly position an a-p polarity cue.

Search, capture and signal: games microtubules and centrosomes play.

Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.

Adherens junctions inhibit asymmetric division in the Drosophila epithelium.

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. Here we show that by disrupting adherens junctions we can convert the symmetric epithelial division into asymmetric division. We further confirm that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrate that both APC and microtubule-associated EB1 homologues are required for the symmetric epithelial division along the planar axis. Our results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions.

Apical, lateral, and basal polarization cues contribute to the development of the follicular epithelium during Drosophila oogenesis.

Analysis of the mechanisms that control epithelial polarization has revealed that cues for polarization are mediated by transmembrane proteins that operate at the apical, lateral, or basal surface of epithelial cells. Whereas for any given epithelial cell type only one or two polarization systems have been identified to date, we report here that the follicular epithelium in Drosophila ovaries uses three different polarization mechanisms, each operating at one of the three main epithelial surface domains. The follicular epithelium arises through a mesenchymal-epithelial transition. Contact with the basement membrane provides an initial polarization cue that leads to the formation of a basal membrane domain. Moreover, we use mosaic analysis to show that Crumbs (Crb) is required for the formation and maintenance of the follicular epithelium. Crb localizes to the apical membrane of follicle cells that is in contact with germline cells. Contact to the germline is required for the accumulation of Crb in follicle cells. Discs Lost (Dlt), a cytoplasmic PDZ domain protein that was shown to interact with the cytoplasmic tail of Crb, overlaps precisely in its distribution with Crb, as shown by immunoelectron microscopy. Crb localization depends on Dlt, whereas Dlt uses Crb-dependent and -independent mechanisms for apical targeting. Finally, we show that the cadherin-catenin complex is not required for the formation of the follicular epithelium, but only for its maintenance. Loss of cadherin-based adherens junctions caused by armadillo (beta-catenin) mutations results in a disruption of the lateral spectrin and actin cytoskeleton. Also Crb and the apical spectrin cytoskeleton are lost from armadillo mutant follicle cells. Together with previous data showing that Crb is required for the formation of a zonula adherens, these findings indicate a mutual dependency of apical and lateral polarization mechanisms.