Sensory Organ Precursor Cells Research Articles

Numb and Alzheimer’s Disease: The Current Picture

Twenty-three years ago, numb was identified as a critical regulator in Drosophila sensory organ precursor cell asymmetric divisions. Beyond the recently recognized role in carcinogenesis, Numb seems to be important in Alzheimer’s disease. This assertion comes from the involvement in various processes such as synapse morphogenesis, amyloid precursor protein trafficking, notch signaling, and neurogenesis. The purpose of the present mini-review is to provide the current picture of Numb’s participation in mechanisms underlying Alzheimer’s disease pathogenesis and emphasize potential aspects for future research.

Complexity at large

Complexity at large

Live-cell Imaging of Sensory Organ Precursor Cells in Intact <em>Drosophila</em> Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact <em>Drosophila</em> Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway. Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Asymmetric cell division in the morphogenesis of Drosophila melanogaster macrochaetae

Asymmetric cell division (ACD) is the basic process which creates diversity in the cells of multicellular organisms. As a result of asymmetric cell division, daughter cells acquire the ability to differentiate and specialize in a given direction, which is different from that of their parent cells and from each other. This type of division is observed in a wide range of living organisms from bacteria to vertebrates. It has been shown that the molecular-genetic control mechanism of ACD is evolutionally conservative. The proteins involved in the process of ACD in different kinds of animals have a high degree of homology. Sensory organs--setae (macrochaetae)--of Drosophila are widely used as a model system for studying the genetic control mechanisms of asymmetric division. Setae located in an orderly manner on the head and body of the fly play the role of mechanoreceptors. Each of them consists of four specialized cells--offspring of the only sensory organ precursor cell (SOPC), which differentiates from the imaginal wing disc at the larval stage of the late third age. The basic differentiation and further specialization of the daughter cells of SOPC is an asymmetric division process. In this summary, experimental data on genes and their products controlling asymmetric division of SOPC and daughter cells, and also the specialization of the latter, have been systemized. The basic mechanisms which determine the time cells enter into asymmetric mitosis and which provides the structural characteristics of the asymmetric division process--the polar distribution of protein determinants Numb and Neuralized--the orientation of the mitotic spindle in relation to these determinants, and the uneven segregation of the determinants into the daughter cells that determines the direction of their development have been discussed.

Intertissue Mechanical Stress Affects Frizzled-Mediated Planar Cell Polarity in the Drosophila Notum Epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features.

Drosophila mechanoreceptors as a model for studying asymmetric cell division

Asymmetric cell division (ACD) is one of the processes creating the overall diversity of cell types in multicellular organisms. The essence of this process is that the daughter cells exit from it being different from both the parental cell and one another in their ability to further differentiation and specialization. The large bristles (macrochaetae) that are regularly arranged on the surface of the Drosophila adult function as mechanoreceptors, and since their development requires ACD, they have been extensively used as a model system for studying the genetic control of this process. Each macrochaete is composed of four specialized cells, the progeny resulting from several ACDs from a single sensory organ precursor (SOP) cell, which differentiates from the ectodermal cells of the wing imaginal disc in the third-instar larva and pupa. In this paper we review the experimental data on the genes and their products controlling the ACDs of the SOP cell and its daughter cells, and their further specialization. We discuss the main mechanisms determining the time when the cell enters ACD, as well as the mechanisms providing for the structural characteristics of asymmetric division, namely, polar distribution of protein determinants (Numb and Neuralized), orientation of the division spindle relative to these determinants, and unequal segregation of the determinants specifying the direction of daughter cell development.

P94. Programmed cell death controls spacing pattern of Drosophila sensory bristles

Bristles on the thorax of the adult fly, called microchaete, are the external sensory organs and regularly spaced. The development of microchaete is well studied as the simple model to investigate the neural pattern formation. The bristle pattern is mainly mediated by the expression of achaete-scute proneural genes and the Notch-mediated lateral inhibition. Epidermis with strong expression of achaete become sensory organ precursor cells (SOPs) and SOPs start to suppress the surrounding epidermal cells to become SOPs via Notch/Delta signaling. By using the live-imaging analysis, we observed that SOPs emerge asynchronously and most of them develop properly. However about 20% of SOPs did not differentiate to sensory organs. These SOPs tend to locate in the ectopic positions, and the time-course of cell division is different compared with normal SOPs. They stop to differentiate after the expression of neural gene, neuralized and most of these ectopic SOPs are eliminated by caspase-dependent cell death at 2-cell stage, and finally proper patterning is achieved. Our results suggest that in the process of spacing pattern formation, programmed cell death functions to eliminate the SOPs that appear ectopically and contributes to the proper pattern formation of sensory bristles.

Notch Signaling: Filopodia Dynamics Confer Robustness

Though usually thought of as mediating communication between adjacent cells, Notch-Delta signaling can take place over a longer range through cellular processes known as 'filopodia'. A recent study shows how the dynamics of filopodia can confer robustness to Notch-Delta dependent patterning.

Error Minimization in Lateral Inhibition Circuits

The pattern of the sensory bristles in the fruit fly Drosophila is remarkably reproducible. Each bristle arises from a sensory organ precursor (SOP) cell that is selected, through a lateral inhibition process, from a cluster of proneural cells. Although this process is well characterized, the mechanism ensuring its robustness remains obscure. Using probabilistic modeling, we defined the sources of error in SOP selection and examined how they depend on the underlying molecular circuit. We found that rapid inhibition of the neural differentiation of nonselected cells, coupled with high cell-to-cell variability in the timing of selection, is crucial for accurate SOP selection. Cell-autonomous interactions (cis interactions) between the Notch receptor and its ligands Delta or Serrate facilitate accurate SOP selection by shortening the effective delay between the time when the inhibitory signal is initiated in one cell and the time when it acts on neighboring cells, suggesting that selection relies on competition between cis and trans interactions of Notch with its ligands. The cis interaction model predicts that the increase in ectopic SOP selections observed with reduced Notch abundance can be compensated for by reducing the abundance of the Notch ligands Delta and Serrate. We validated this prediction experimentally by quantifying the frequency of ectopic bristles in flies carrying heterozygous null mutations of Notch, Delta, or Serrate or combinations of these alleles. We propose that susceptibility to errors distinguishes seemingly equivalent designs of developmental circuits regulating pattern formation.

Dynamic intracellular distribution of Notch during activation and asymmetric cell division revealed by functional fluorescent fusion proteins

Notch signaling mediates intercellular communication required for cell fate specification. Because the majority of Notch proteins exist as an intracellular pool, information on intracellular trafficking of Notch is essential for understanding its regulation. To observe trafficking of Notch protein in vivo, we constructed a Notch protein with green fluorescent protein (GFP) inserted into the intracellular domain. This construct rescued the neurogenic phenotype of Notch mutant embryos and caused the typical Notch gain-of-function phenotype when over-expressed in the wing imaginal disc. Using this and similarly constructed Notch-mCherry fusion, we showed rapid spatio-temporal dynamics of Notch in endosomes. Kinetic analysis revealed that activation of Notch-GFP by ectodomain shedding causes fast and unrestricted nuclear translocation of Notch intracellular domain. During asymmetric cell division of sensory organ precursor cells, distribution of Notch-GFP showed transient asymmetry at the interface of daughter cells, confirming a possible site of regulation that biases Notch signaling level among daughter cells.

Atonal, Senseless, and Abdominal-A regulate rhomboid enhancer activity in abdominal sensory organ precursors

The atonal ( ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid ( rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. We previously identified a rho enhancer (RhoBAD) that is active within a subset of abdominal SOP cells to induce larval oenocytes and showed that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor. Here, we show that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, we propose a model for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression.

Neuralized Promotes Basal to Apical Transcytosis of Delta in Epithelial Cells

Notch receptors mediate short-range signaling controlling many developmental decisions in metazoans. Activation of Notch requires the ubiquitin-dependent endocytosis of its ligand Delta. How ligand endocytosis in signal-sending cells regulates receptor activation in juxtaposed signal-receiving cells remains largely unknown. We show here that a pool of Delta localizes at the basolateral membrane of signal-sending sensory organ precursor cells in the dorsal thorax neuroepithelium of Drosophila and that Delta is endocytosed in a Neuralized-dependent manner from this basolateral membrane. This basolateral pool of Delta is segregated from Notch that accumulates apically. Using a compartimentalized antibody uptake assay, we show that murine Delta-like 1 is similarly internalized by mNeuralized2 from the basolateral membrane of polarized Madin-Darby canine kidney cells and that internalized ligands are transcytosed to the apical plasma membrane where mNotch1 accumulates. Thus, endocytosis of Delta by Neuralized relocalizes Delta from the basolateral to the apical membrane domain. We speculate that this Neuralized-dependent transcytosis regulates the signaling activity of Delta by relocalizing Delta from a membrane domain where it cannot interact with Notch to another membrane domain where it can bind and activate Notch.

Laser Microdissection of Sensory Organ Precursor Cells of Drosophila Microchaetes

BackgroundIn Drosophila, each external sensory organ originates from the division of a unique precursor cell (the sensory organ precursor cell or SOP). Each SOP is specified from a cluster of equivalent cells, called a proneural cluster, all of them competent to become SOP. Although, it is well known how SOP cells are selected from proneural clusters, little is known about the downstream genes that are regulated during SOP fate specification.Methodology/Principal FindingsIn order to better understand the mechanism involved in the specification of these precursor cells, we combined laser microdissection, toisolate SOP cells, with transcriptome analysis, to study their RNA profile. Using this procedure, we found that genes that exhibit a 2-fold or greater expression in SOPs versus epithelial cells were mainly associated with Gene Ontology (GO) terms related with cell fate determination and sensory organ specification. Furthermore, we found that several genes such as pebbled/hindsight, scabrous, miranda, senseless, or cut, known to be expressed in SOP cells by independent procedures, are particularly detected in laser microdissected SOP cells rather than in epithelial cells.Conclusions/SignificanceThese results confirm the feasibility and the specificity of our laser microdissection based procedure. We anticipate that this analysis will give new insight into the selection and specification of neural precursor cells.

Numb Independently Antagonizes Sanpodo Membrane Targeting and Notch Signaling in Drosophila Sensory Organ Precursor Cells

In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure-function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.

Error Minization in Lateral Inhibition Circuits

D ow nladed from The pattern of the sensory bristles in the fruit fly Drosophila is remarkably reproducible. Each bristle arises from a sensory organ precursor (SOP) cell that is selected, through a lateral inhibition process, from a cluster of proneural cells. Although this process is well characterized, the mechanism ensuring its robustness remains obscure. Using probabilistic modeling, we defined the sources of error in SOP selection and examined how they depend on the underlying molecular circuit. We found that rapid inhibition of the neural differentiation of nonselected cells, coupled with high cell-to-cell variability in the timing of selection, is crucial for accurate SOP selection. Cell-autonomous interactions (cis interactions) between the Notch receptor and its ligands Delta or Serrate facilitate accurate SOP selection by shortening the effective delay between the time when the inhibitory signal is initiated in one cell and the time when it acts on neighboring cells, suggesting that selection relies on competition between cis and trans interactions of Notch with its ligands. The cis interaction model predicts that the increase in ectopic SOP selections observed with reduced Notch abundance can be compensated for by reducing the abundance of the Notch ligands Delta and Serrate. We validated this prediction experimentally by quantifying the frequency of ectopic bristles in flies carrying heterozygous null mutations of Notch, Delta, or Serrate or combinations of these alleles. We propose that susceptibility to errors distinguishes seemingly equivalent designs of developmental circuits regulating pattern formation. tt

The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development

Mutations in either of the genes encoding the tuberous sclerosis complex (TSC), TSC1 and TSC2, result in a multisystem tumor disorder characterized by lesions with unusual lineage expression patterns. How these unusual cell-fate determination patterns are generated is unclear. We therefore investigated the role of the TSC in the Drosophila external sensory organ (ESO), a classic model of asymmetric cell division. In normal development, the sensory organ precursor cell divides asymmetrically through differential regulation of Notch signaling to produce a pIIa and a pIIb cell. We report here that inactivation of Tsc1 and overexpression of the Ras homolog Rheb each resulted in duplication of the bristle and socket cells, progeny of the pIIa cell, and loss of the neuronal cell, a product of pIIb cell division. Live imaging of ESO development revealed this cell-fate switch occurred at the pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often found in tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target gene activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2 mutations exhibited TSC2- and Rheb-dependent Notch activation. Finally, inhibition of Notch signaling using a gamma-secretase inhibitor suppressed proliferation of Tsc2-null rat cells in a xenograft model. Together, these data indicate that the TSC and Rheb regulate Notch-dependent cell-fate decision in Drosophila and Notch activity in mammalian cells and that Notch dysregulation may underlie some of the distinctive clinical and pathologic features of TSC.

S07-04 Sara endosomes during asymmetric cell division

Recent studies have uncovered a key role of endocytosis during Notch signalling after the asymmetric division of the fly sensory organ precursor cells (SOP): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, the pIIb. Here we show a novel mechanism of directional signalling based on the trafficking of Delta/Notch molecules already internalized in the SOP and targeted subsequently to the pIIa daughter. We show that, in the SOP, internalized Delta and Notch traffic to an endosome characterized by the localization of the endosomal protein Sara. During SOP division, Sara endosomes and Notch/Delta therein move first to the central spindle and then to the pIIa cell in a process mediated by the Par-complex. Subsequently, in pIIa (but not pIIb), Notch appears cleaved in Sara endosomes in a process that requires gamma-secretase and Delta internalization implying that the intracellular Notch tail has been released to mediate transcription of Notch target genes. We thus uncovered two phases during biased Notch signalling: (i) in the mother, receptor molecules internalized into Sara endosomes are targeted to the pIIa, thereby increasing the level of Notch signaling in pIIa and decreasing it in pIIb and, (ii) subsequently, biased signaling is also mediated by endocytic trafficking leading to Sanpodo downregulation and Delta recycling/activation in the pIIb daughter cell.

Genome Engineering-Based Analysis of Bearded Family Genes Reveals Both Functional Redundancy and a Nonessential Function in Lateral Inhibition in Drosophila

Lateral inhibition mediated by Notch receptor signaling regulates the determination of sensory organ precursor cells (SOPs) in Drosophila. The selection of SOPs from proneural cluster cells appears to rely on a negative feedback loop linking activation of the Notch receptor to downregulation of its ligand Delta within each cell. The molecular basis of this regulatory feedback mechanism is not known. Here, we have tested the role of the Bearded (Brd) family genes in this process. The Drosophila genome encodes eight Brd family members that interact with the E3 ubiquitin ligase Neuralized (Neur) and act as inhibitors of Neur-mediated Delta signaling. Genome engineering technologies were used to create specific deletions of all eight Brd family genes. We find that the Brd family genes malpha, m4, and m6 encoded by the Enhancer of split Complex (E(spl)-C) are dispensable for Drosophila development and that deletion of the five Brd family genes encoded by the Brd Complex only reduces viability. However, deletion of all Brd family genes results in embryonic lethality. Additionally, the malpha, m4, and m6 genes act redundantly with the other five Brd family genes to spatially restrict Notch activation in stage 5 embryos. These data reveal that the Brd family genes have an essential but redundant activity. While the activity of all eight Brd genes appears to be dispensable for SOP determination, clone border studies indicate that both the relative activity levels of Neur and Brd family members influence competition for the SOP fate during lateral inhibition. We propose that inhibition of Neur-Delta interaction by Brd family members is part of the feedback loop that underlies lateral inhibition in Drosophila.

Both inhibition and activation of Notch signaling rely on a conserved Neuralized-binding motif in Bearded proteins and the Notch ligand Delta

Lateral inhibition is one of the key functions of Notch signaling during animal development. In the proneural clusters that give rise to Drosophila mechanosensory bristles, Delta (Dl) ligand in the sensory organ precursor (SOP) cell is targeted for ubiquitination by the E3 ligase Neuralized (Neur), resulting in activation of Dl's capacity to signal to the Notch receptor on neighboring cells. The cells that receive this signal activate a genetic program that suppresses their SOP fate potential, insuring that only a single SOP develops within each cluster. Using multiple lines of investigation, we provide evidence that members of the Bearded family of proteins (BFMs) inhibit Dl activation in non-SOP cells by binding to Neur and preventing it from interacting with Dl. We show that this activity of BFMs is dependent on the conserved NXXN motif, and report the unexpected finding that several BFMs include multiple functional copies of this motif. We find that a conserved NXXN motif in the intracellular domain of Dl is responsible for its interaction with Neur, indicating direct competition between Dl and BFMs for binding to Neur, and we show that Neur-dependent endocytosis of Dl requires the integrity of its NXXN motif. Our results illuminate the mechanism of an important regulatory event in Notch signaling that appears to be conserved between insects and crustaceans.