Drosophila E-cadherin regulates the orientation of asymmetric cell division in the sensory organ lineage

BACKGROUND: Generation of cell-fate diversity in Metazoan depends in part on asymmetric cell divisions in which cell-fate determinants are asymmetrically distributed in the mother cell and unequally partitioned between daughter cells. The polarization of the mother cell is a prerequisite to the unequal segregation of cell-fate determinants. In the Drosophila bristle lineage, two distinct mechanisms are known to define the axis of polarity of the pI and pIIb cells. Frizzled (Fz) signaling regulates the planar orientation of the pI division, while Inscuteable (Insc) directs the apical-basal polarity of the pIIb cell. The orientation of the asymmetric division of the pIIa cell is identical to the one of its mother cell, the pI cell, but, in contrast, is regulated by an unknown Insc- and Fz-independent mechanism. RESULTS: DE-Cadherin-Catenin complexes are shown to localize at the cell contact between the two cells born from the asymmetric division of the pI cell. The mitotic spindle of the dividing pIIa cell rotates to line up with asymmetrically localized DE-Cadherin-Catenin complexes. While a complete loss of DE-Cadherin function disrupts the apical-basal polarity of the epithelium, both a partial loss of DE-Cadherin function and expression of a dominant-negative form of DE-Cadherin affect the orientation of the pIIa division. Furthermore, expression of dominant-negative DE-Cadherin also affects the position of Partner of Inscuteable (Pins) and Bazooka, two asymmetrically localized proteins known to regulate cell polarity. These results show that asymmetrically distributed Cad regulates the orientation of asymmetric cell division. CONCLUSIONS: We describe a novel mechanism involving a specialized Cad-containing cortical region by which a daughter cell divides with the same orientation as its mother cell.     

Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. Here we have observed the asymmetric divisions that give rise to these cells in the developing pupae using green fluorescent protein fusion proteins. We find that spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, we find that two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorientates to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, we propose that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila.     

Cell lineage analysis of the Drosophila peripheral nervous system

The peripheral nervous system (PNS) of Drosophila provides a very well-characterized model system for studying the genes involved in basic processes of neurogenesis. Because of its simplicity and stereotyped pattern, each cell of the PNS can be individually identified and the phenotypic consequences of mutations can be studied in detail. Thus, some of the genetic mechanisms leading to the formation of type I sensory organs, the external, bristle-type sensory organs (es), and the internal, stretch-receptive chordotonal organs (ch) have been elucidated. Each sensory organ seems to be generated by a stereotyped pattern of cell division of individual ectodermal precursor cells. Recent advances in cell lineage analysis of the PNS have provided a detailed picture of almost all the lineages in the PNS, including those giving rise to the type II sensory neurons, also known as multiple dendritic (md) neurons. This knowledge will be instrumental in the precise characterization of the phenotypes associated with mutations in known and new genes and their interactions which determine cell fate decisions during neurogenesis. Here, we describe and compare three recently developed methods by which cell lineages have been assessed: single cell transplantation, bromodeoxyuridine (BrdU) incorporation studies, and the flp/FRT recombinase system from yeast. In the light of a more complete knowledge of the PNS lineages, we will discuss the effects of known mutations that alter neuronal cell fates. © 1996 Wiley-Liss, Inc.     

Flamingo controls the planar polarity of sensory bristles and asymmetric division of sensory organ precursors in Drosophila.

The sensory bristles of the fruit fly Drosophila are organized in a polarized fashion such that bristles on the thorax point posteriorly. These bristles are derived from asymmetric division of sensory organ precursors (SOPs). The Numb protein, which is localized asymmetrically in a cortical crescent in each SOP, segregates into only one of the two daughter cells during cell division, thereby conferring distinct fates to the daughter cells [1] [2]. In neuroblasts, establishment of apical-basal polarity by the protein Inscuteable is crucial for orienting asymmetric division, but this is not the case for division of SOPs [3]. Instead, the Frizzled (Fz) protein mediates a planar polarity signal that controls the anteroposteriorly oriented first division (pl) of SOPs [4]. Here, we report that Flamingo (Fmi), a seven-transmembrane cadherin [5], controls the planar polarity of sensory bristles and the orientation of the SOP pl division. Both the loss of function and overexpression of fmi disrupted bristle polarity. During mitosis of the SOP, the axis of the pl division and the positioning of the Numb crescent were randomized in the absence of Fmi activity. Overexpression of Fmi and Fz caused similar effects. The dependence of proper Fmi localization on Fz activity suggests that Fmi functions downstream of Fz in controlling planar polarity. We also present evidence suggesting that Fz also functions in the Wingless pathway to pattern sensory organs.     

A mis-expression study of factors affecting Drosophila PNS cell identity

Drosophila PNS sense organs arise from single sensory organ precursor (SOP) cells through a series of asymmetric divisions. In a mis-expression screen for factors affecting PNS development, we identified string and dappled as being important for the proper formation of adult external sensory (ES) organs. string is a G2 regulator. dappled has no described function but is implicated in tumorigenesis. The mis-expression effect from string was analysed using timed over expression during adult ES-organ and, for comparison, embryonic Chordotonal (Ch) organ formation. Surprisingly, string mis-expression prior to SOP division gave the greatest effect in both systems. In adult ES-organs, this lead to cell fate transformations producing structural cells, whilst in the embryo organs were lost, hence differences within the lineages exist. Mis-expression of dappled, lead to loss and duplications of entire organs in both systems, potentially affecting SOP specification, in addition to affecting neuronal guidance.     

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.     

Lethal giant larvae acts together with numb in notch inhibition and cell fate specification in the Drosophila adult sensory organ precursor lineage

The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. We report here that, in contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages.     

Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling

Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.     

Drosophila Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval PNS. We report a role of scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis we find that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an “off-centred” manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality.     

A dual function of phyllopod in Drosophila external sensory organ development: cell fate specification of sensory organ precursor and its progeny

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from a proneural cluster, and undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. We show that phyllopod (phyl), previously identified to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters, leading to the fate transformation of neurons and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. We further show that seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. It has been shown previously that Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. We further demonstrate that phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development.     

A hidden program in Drosophila peripheral neurogenesis revealed: fundamental principles underlying sensory organ diversity

How is cell fate diversity reliably achieved during development? Insect sensory organs have been a favorable model system for investigating this question for over 100 years. They are constructed using defined cell lineages that generate a maximum of cell diversity with a minimum number of cell divisions, and display tremendous variety in their morphologies, constituent cell types, and functions. An unexpected realization of the past 5 years is that very diverse sensory organs in Drosophila are produced by astonishingly similar cell lineages, and that their diversity can be largely attributed to only a small repertoire of developmental processes. These include changes in terminal cell differentiation, cell death, cell proliferation, cell recruitment, cell-cell interactions, and asymmetric segregation of cell fate determinants during mitosis. We propose that most Drosophila sensory organs are built from an archetypal lineage, and we speculate about how this stereotyped pattern of cell divisions may have been built during evolution.     

Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.     

Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division

Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior-posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.     

Unequal segregation of Neuralized biases Notch activation during asymmetric cell division

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). We report that the E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. We propose that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling.     

Mother-daughter precursor cell fate transformation after Cdc2 down-regulation in the Drosophila bristle lineage

The Drosophila bristle lineage is an excellent system in which to study how cell cycle and fate determination are synchronized in invariant cell lineages. In this model, five different cells arise from a single precursor cell, pI, after four asymmetric cell divisions. Cell diversity is achieved by the asymmetric segregation of cell determinants, such as Numb and Neuralized (Neur), resulting in differential activation of the Notch (N) pathway. We show that down-regulation of Cdc2, by over-expressing Tribbles, Dwee1, and Dmyt1 (three negative regulators of Cdc2) or by using thermo-sensitive Cdc2 mutant flies, delayed pI mitosis, and altered the polarity and the number of subsequent cell divisions. These modifications were associated with a mother-daughter cell fate transformation as the pI cell acquired the identity of the secondary precursor cell, pIIb. This type of change in cell identity only occurred when the N signaling pathway was inactive since ectopic N signaling transformed pI to pIIa-progeny fate. These transformations in cell identity suggest that, although synchronized, cell cycle and fate determination are independent phenomena in the bristle lineage.     

Drosophila neural progenitor polarity and asymmetric division

In the Drosophila embryonic central nervous system, the neural precursor cells called neuroblasts undergo a number of asymmetric divisions along the apical-basal axis to give rise to different daughter cells of distinct fates. This review summarizes recent progress in understanding the mechanisms of these asymmetric cell divisions. We discuss proteins that are localized at distinct domains of cortex in the neuroblasts and their role in generating asymmetry. We also review uniformly cortical localized factors and actin cytoskeleton-associated motor proteins with regard to their potential role to serve as a link between distinct cortical domains in the neuroblasts. In this review, asymmetric divisions of sensory organ precursor and larval neuroblasts are also briefly discussed.     

Binary cell death decision regulated by unequal partitioning of Numb at mitosis

An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. We describe here a Drosophila sensory organ lineage that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. Our study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner.     

A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila

BACKGROUND: In the fruit fly Drosophila, the Inscuteable protein localises to the apical cell cortex in neuroblasts and directs both the apical-basal orientation of the mitotic spindle and the basal localisation of the protein determinants Numb and Prospero during mitosis. Asymmetric localisation of Inscuteable is initiated during neuroblast delamination by direct binding to Bazooka, an apically localised protein that contains protein-interaction motifs known as PDZ domains. How apically localised Inscuteable directs asymmetric cell divisions is unclear. RESULTS: A novel 70 kDa protein called Partner of Inscuteable (Pins) and a heterotrimeric G-protein alpha subunit were found to bind specifically to the functional domain of Inscuteable in vivo. The predicted sequence of Pins contained tetratrico-peptide repeats (TPRs) and motifs implicated in binding Galpha proteins. Pins colocalised with Inscuteable at the apical cell cortex in interphase and mitotic neuroblasts. Asymmetric localisation of Pins required both Inscuteable and Bazooka. In epithelial cells, which do not express inscuteable, Pins was not apically localised but could be recruited to the apical cortex by ectopic expression of Inscuteable. In pins mutants, these epithelial cells were not affected, but neuroblasts showed defects in the orientation of their mitotic spindle and the basal asymmetric localisation of Numb and Miranda during metaphase. Although localisation of Inscuteable in pins mutants was initiated correctly during neuroblast delamination, Inscuteable became homogeneously distributed in the cytoplasm during mitosis. CONCLUSIONS: Pins and Inscuteable are dependent on each other for asymmetric localisation in delaminated neuroblasts. The binding of Pins to Galpha protein offers the intriguing possibility that Inscuteable and Pins might orient asymmetric cell divisions by localising or locally modulating a heterotrimeric G-protein signalling cascade at the apical cell cortex.     

Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.