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1.  A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot MARCM 
PLoS Biology  2010;8(8):e1000461.
Labeling every neuron in a lineage in the fruit fly olfactory system reveals that every cell is born with a pre-determined cell fate that is invariant and dependent upon neuron birth order
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.
Author Summary
A brain consists of numerous, potentially individually unique neurons that derive from a limited number of progenitors. It has been shown in various model organisms that specific neurons arise in a lineage made by a repeatedly renewing progenitor at specific times of development. However, except in the worm C. elegans, the stereotype of neural development has never been examined in sufficient detail to account for every single neuron derived from a common progenitor. Here we applied a sophisticated genetic mosaic system to mark single neurons in the adult Drosophila brain and simultaneously reveal in which lineage a targeted neuron had arisen and when along the lineage it was made. We have identified each neuron in a lineage of olfactory projection neurons. There are a remarkable 40 types of neurons within this lineage born over two epochs. Strikingly, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death, such that every neuron type has a unique and invariant cell count. Sequencing an entire neuronal lineage provides definitive evidence for origin-dependent neuron type specification. It further permits a systematic characterization of neuron types for comprehensive circuitry mapping.
PMCID: PMC2927434  PMID: 20808769
2.  Identification of a new stem cell population that generates Drosophila flight muscles 
eLife  2014;3:e03126.
How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development.
eLife digest
Muscle tissues must grow and change to accommodate the needs of an animal at various stages in its life. For example, fruit flies begin life as larvae and their muscles must help them move their soft bodies. Later, when the flies mature into adults, the muscles must provide power for flight and support for the insects' external skeletons.
Like other animal tissues, muscles develop from non-specialized stem cells which at first have the potential to become almost any cell type, but later change to become more specialized. Studies of fruit flies, in particular, have yielded insights on how pools of stem cell are created and regulated. Fruit flies are small and easier to study than larger organisms, and as a result, scientists have learned a lot about their genetics and cell biology. Gunage et al. have now identified the stem cell pools that develop into flight muscle tissue, and found that these cells were set aside for the muscles when the fruit fly embryo was still developing.
Fruit flies have large forewings that power flight, and small modified hindwings (called halteres) that help the insect to balance when flying. Gunage et al. reveal that a small, but similar, number of cells are set aside to make both both the tiny muscles that will move the halteres and the much larger flight muscles that move the forewings. However, the cells that contribute to the flight muscles divide to give far more muscle progenitor cells than their haltere counterparts, and make a couple of thousand cells that eventually fuse to form muscle fibers.
Gunage et al. looked at how the flight muscle progenitors multiplied by genetically engineering some of the stem cells in fruit fly larvae so that when each cell divided, its two daughter cells would fluoresce with different colors. One daughter cell would glow green and the other glow red. Gunage et al. found that at first the cells multiply equally, with half the new cells coming from a ‘red’ stem cell and the other half from a ‘green’ cell—meaning that the number of cells increases exponentially. Later, the balance shifted so that either more red cells than green cells were produced, or vice versa. This results in a ‘linear’ increase in number of muscle progenitor cells. Furthermore, Gunage et al. identified the proteins that orchestrate the switch from equal to unequal multiplying of these cells at the different times points in the fruit flies’ development.
The next challenge is to see if these stem cells that form the muscles are also available for repair of mature muscle tissue after it is damaged. If this is so, these stems cells might perform a similar function to muscle satellite cells, which are found in the mature muscles of mammals and other vertebrates.
PMCID: PMC4171707  PMID: 25135939
stem cells; muscles; numb; Wnt; Notch; niche; D. melanogaster
3.  Lineage Analysis of Drosophila Lateral Antennal Lobe Neurons Reveals Notch-Dependent Binary Temporal Fate Decisions 
PLoS Biology  2012;10(11):e1001425.
A high-resolution neuronal lineage analysis in the Drosophila antennal lobe reveals the complexity of lineage development and Notch signaling in cell fate specification.
Binary cell fate decisions allow the production of distinct sister neurons from an intermediate precursor. Neurons are further diversified based on the birth order of intermediate precursors. Here we examined the interplay between binary cell fate and birth-order-dependent temporal fate in the Drosophila lateral antennal lobe (lAL) neuronal lineage. Single-cell mapping of the lAL lineage by twin-spot mosaic analysis with repressible cell markers (ts-MARCM) revealed that projection neurons (PNs) and local interneurons (LNs) are made in pairs through binary fate decisions. Forty-five types of PNs innervating distinct brain regions arise in a stereotyped sequence; however, the PNs with similar morphologies are not necessarily born in a contiguous window. The LNs are morphologically less diverse than the PNs, and the sequential morphogenetic changes in the two pairs occur independently. Sanpodo-dependent Notch activity promotes and patterns the LN fates. By contrast, Notch diversifies PN temporal fates in a Sanpodo-dispensable manner. These pleiotropic Notch actions underlie the differential temporal fate specification of twin neurons produced by common precursors within a lineage, possibly by modulating postmitotic neurons' responses to Notch-independent transcriptional cascades.
Author Summary
The Drosophila brain develops from a limited number of neural stem cells that produce a series of ganglion mother cells (GMCs) that divide once to produce a pair of neurons in a defined order, termed a neuronal lineage. Here, we provide a detailed lineage map for the neurons derived from the Drosophila lateral antennal lobe (lAL) neuroblast. The lAL lineage consists of two distinct hemilineages, generated through differential Notch signaling in the two GMC daughters, to produce one projection neuron (PN) paired with a local interneuron (LN). Both hemilineages yield distinct cell types in the same sequence, although the temporal identity (birth-order-dependent fate) changes are regulated independently between projection neurons and local interneurons, such that a series of analogous local interneurons may co-derive with different projection neurons and vice versa. We also find that Notch signaling can transform a class of nonantennal lobe projection neurons into antennal lobe projection neurons. These findings suggest that Notch signaling not only modulates temporal fate but itself plays a role in the distinction of antennal lobe versus nonantennal lobe neurons.
PMCID: PMC3502534  PMID: 23185131
4.  Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway 
Cell Cycle  2011;10(23):4110-4118.
Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs) that encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here, we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway, including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs and place Drosophila endocytic nTSGs into a network that involves two major signaling pathways implicated in oncogenesis.
PMCID: PMC3272291  PMID: 22101275
Drosophila; endocytic tumor suppressor; Yki; JNK; Tsg101; AP-2; Hippo
5.  Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal 
Developmental biology  2009;330(2):399-405.
Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis.
PMCID: PMC2728501  PMID: 19374896
6.  Interaction with Tsg101 Is Necessary for the Efficient Transport and Release of Nucleocapsids in Marburg Virus-Infected Cells 
PLoS Pathogens  2014;10(10):e1004463.
Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARVPSAPmut). rMARVPSAPmut was attenuated by up to one log compared with recombinant wild-type MARV (rMARVwt), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARVPSAPmut-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARVwt-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARVwt-infected cells and was co-transported together with nucleocapsids. In contrast, rMARVPSAPmut nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.
Author Summary
Marburg virus (MARV) is endemic in central Africa and causes hemorrhagic fever in humans and non-human primates, with high lethality. Presumably, the disease severity primarily depends on the response of host-cell factors interacting with viral proteins. We generated a recombinant MARV encoding an NP with a mutated PSAP late domain motif, which has previously been shown to mediate interaction with the cellular ESCRT protein Tsg101. We found that the PSAP-mediated interaction with Tsg101 was important at several steps of MARV assembly before viral fission. First, the egress of mature rMARVPSAPmut nucleocapsids from viral inclusions was inhibited. Second, actin-driven transport of rMARVPSAPmut nucleocapsids was impaired, displaying significantly shortened trajectories and reduced movement in the cell periphery. Third, rMARVPSAPmut nucleocapsids accumulated in cell periphery, and the number of filopodia-associated nucleocapsids decreased, indicating that rMARVPSAPmut nucleocapsids were defective to enter filopodia, the major budding sites of MARV. These defects resulted in the attenuated growth of rMARVPSAPmut. Interestingly, IQGAP1, an actin cytoskeleton regulator which interacts with Tsg101, was also recruited to nucleocapsids in dependence of the PSAP late domain. Thus, the interaction of NP with Tsg101 not only impacts viral budding at the plasma membrane but also nucleocapsid transport through the cytoplasm.
PMCID: PMC4199773  PMID: 25330247
7.  Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted gastrulation 
Developmental biology  2008;323(1):6-18.
Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2;Tsg and Cv2;Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.
PMCID: PMC2647368  PMID: 18789316
BMP; Crossveinless-2; Chordin; Twisted Gastrulation; Tolloid; vertebra; morphogenetic field; cartilage; pattern formation
8.  Novel mechanism for mesenchymal stem cells in attenuating peritoneal adhesion: accumulating in the lung and secreting tumor necrosis factor α-stimulating gene-6 
We previously found that mesenchymal stem cells (MSCs) injected intravenously could attenuate peritoneal adhesion by secreting tumor necrosis alpha-stimulating gene (TSG)-6, while MSCs injected intraperitoneally could not. However, the underlying mechanism remains unclear. This study was designed to investigate the means by which MSCs exert their effects.
Rat bone marrow-derived MSCs/red fluorescent protein (RFP) were injected either intraperitoneally or intravenously into Sprague-Dawley (SD) rats at different time points after peritoneal scraping. Peritoneal adhesions were evaluated macroscopically at day 14 after scraping. The distribution of MSCs injected intraperitoneally or intravenously was traced by two-photon fluorescence confocal imaging and immunofluorescence microscopy. The co-localization of MSCs and macrophages in the lung and the spleen, and the expression of TSG-6 in MSCs trapped in the lung or the spleen were evaluated by immunofluorescence microscopy. The concentration of TSG-6 in serum was evaluated by ELISA. After intravenous injection of TSG-6- small interfering (si) RNA-MSCs, the expression of TSG-6 in MSCs and the concentration of TSG-6 in serum were reevaluated, and peritoneal adhesions were evaluated macroscopically and histologically.
MSCs injected intraperitoneally failed to reduce peritoneal adhesion, and MSCs injected intravenously markedly improved peritoneal adhesion. Two-photon fluorescence confocal imaging showed that MSCs injected intravenously accumulated mainly in the lung, where they remained for seven days, and immunofluorescence microscopy showed few MSCs phagocytosed by macrophages. In contrast, large numbers of MSCs accumulated in the spleen with obvious phagocytosis by macrophages even at 4 hours after intraperitoneal injection. Immunofluorescence microscopy showed that MSCs that accumulated in the lung after intravenous injection could express TSG-6 within 12 hours, but TSG-6-siRNA-MSCs or MSCs accumulated in the spleen after intraperitoneal injection did not. ELISA showed that the concentration of TSG-6 in serum was increased at 4 hours after intravenous injection of MSCs, while there was no increase after injection of TSG-6-siRNA-MSCs or after intraperitoneal injection of MSCs. Moreover, intravenous injection of TSG-6-siRNA-MSCs failed to attenuate peritoneal adhesion.
Our findings suggest that intravenously injected MSCs accumulated in the lung and attenuated peritoneal adhesion by secreting TSG-6, but intraperitoneally injected MSCs were phagocytosed by macrophages in the spleen and failed to attenuate peritoneal adhesion.
PMCID: PMC3580481  PMID: 23217986
9.  Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila 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).
PMCID: PMC3125114  PMID: 21654627
10.  Suppression of Scant Identifies Endos as a Substrate of Greatwall Kinase and a Negative Regulator of Protein Phosphatase 2A in Mitosis 
PLoS Genetics  2011;7(8):e1002225.
Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. Heterozygosity for an endos mutation suppresses this PP2A/polo11 sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwlScant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop.
Author Summary
Progression through mitosis requires the addition of phosphate groups onto specific proteins by enzymes collectively known as mitotic protein kinases. At the end of mitosis, these phosphates are removed by protein phosphatases. Whereas we know quite a lot about the mitotic protein kinases, we know much less about the phosphatases. Here we used the fruit fly Drosophila as a model organism to identify a pathway regulating a phosphatase required for mitotic exit. Using mutations in genes for this pathway in the fly and by depleting levels of corresponding proteins from cultured cells, we established the relationships between the gene products. This has revealed that Greatwall mitotic kinase works in concert with the protein Endos to antagonise Protein Phosphatase 2A (PP2A). Specifically, Greatwall and Endos affect the activity of a particular form of PP2A that is associated with only one of the four different regulatory subunits found in Drosophila. We found that phosphorylation of Endos at a defined position by Greatwall kinase is required for its function. Together this provides genetic evidence that the Greatwall mitotic kinase inhibits the PP2A phosphatase required for mitotic exit thus complementing biochemical experiments using frog eggs and indicating the universality of this mechanism.
PMCID: PMC3154957  PMID: 21852956
11.  Orthodenticle is necessary for survival of a cluster of clonally related dopaminergic neurons in the Drosophila larval and adult brain 
Neural Development  2011;6:34.
The dopaminergic (DA) neurons present in the central brain of the Drosophila larva are spatially arranged in stereotyped groups that define clusters of bilaterally symmetrical neurons. These clusters have been classified according to anatomical criteria (position of the cell bodies within the cortex and/or projection pattern of the axonal tracts). However, information pertaining to the developmental biology, such as lineage relationship of clustered DA neurons and differential cell subtype-specific molecular markers and mechanisms of differentiation and/or survival, is currently not available.
Using MARCM and twin-spot MARCM techniques together with anti-tyrosine hydroxylase immunoreactivity, we have analyzed the larval central brain DA neurons from a developmental point of view and determined their time of birth, their maturation into a DA neurotransmitter phenotype as well as their lineage relationships. In addition, we have found that the homeodomain containing transcription factor Orthodenticle (Otd) is present in a cluster of clonally related DA neurons in both the larval and adult brain. Taking advantage of the otd hypomorphic mutation ocelliless (oc) and the oc2-Gal4 reporter line, we have studied the involvement of orthodenticle (otd) in the survival and/or cell fate specification of these post-mitotic neurons.
Our findings provide evidence of the presence of seven neuroblast lineages responsible for the generation of the larval central brain DA neurons during embryogenesis. otd is expressed in a defined group of clonally related DA neurons from first instar larvae to adulthood, making it possible to establish an identity relationship between the larval DL2a and the adult PPL2 DA clusters. This poses otd as a lineage-specific and differential marker of a subset of clonally related DA neurons. Finally, we show that otd is required in those DA neurons for their survival.
PMCID: PMC3206411  PMID: 21999236
The Journal of biological chemistry  2005;280(35):30899-30908.
We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We show that recombinant full length human TSG-6 (TSG-6Q) and Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin is due to its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation-dependence is due to conformational effects of Ca-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of inter-α-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.
PMCID: PMC1351260  PMID: 16006654
13.  Monozygotic Twins Discordant for Neurofibromatosis 1 
We present monozygotic twins discordant for the autosomal dominant disorder neurofibromatosis type 1 (NF1). The affected twin was diagnosed with NF1 at age 12, based upon accepted clinical criteria for the disorder. Both twins were re-examined at ages 35 and 57, at which times the unaffected twin continued to show no clinical manifestations of NF1. Short tandem repeat marker (STR) genotyping at 10 loci on chromosome 17 and 10 additional loci dispersed across the genome revealed identical genotypes for the twins, confirming their monozygosity. The affected twin has three children, two of whom also have NF1, while the unaffected twin has two children, both unaffected. Using lymphoblastoid, fibroblast, and buccal cell samples collected from both twins and from other family members in three generations, we discovered a pathogenic nonsense mutation in exon 40 of the NF1 gene. This mutation was found in all cell samples from the affected twin and her affected daughter, and in lymphoblastoid and buccal cells but not fibroblasts from the unaffected twin. We also found a novel non-synonymous change in exon 16 of the NF1 gene that was transmitted from the unaffected mother to both twins and co-segregated with the pathogenic mutation in the ensuing generation. All cells from the twins were heterozygous for this apparent exon 16 polymorphism and for single nucleotide polymorphisms (SNPs) within 2.5kb flanking the site of the exon 40 nonsense mutation. This suggests that the NF1 gene of the unaffected twin differed in the respective lymphoblastoid cells and fibroblasts only at the mutation site itself, making post-zygotic mutation leading to mosaicism the most likely mechanism of phenotypic discordance. Although the unaffected twin is a mosaic, the distribution of the mutant allele among different cells and tissues appears to be insufficient to cause overt clinical manifestations of NF1.
PMCID: PMC2830382  PMID: 20186797
Neurofibromatosis type 1; monozygotic twins; discordance; post-zygotic mutation
14.  EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells 
eLife  null;3:e04437.
Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. We found that, in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, epidermal growth factor receptor (EGFR) signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical–basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, we identified a novel connection between EGFR signaling and the cell-polarity regulator liver kinase B1 (LKB1), which indicates that EGFR signals through both the Ras–Raf–MEK–Erk pathway and through the LKB1–AMPK pathway to suppress apical identity. The development of apical–basal polarity is the earliest visible difference between FSCs and their daughters, and our findings demonstrate that the EGFR-mediated regulation of apical–basal polarity is essential for the segregation of stem cell and daughter cell fates.
eLife digest
A stem cell is a special cell that divides to produce another stem cell, plus a cell that goes on to perform a specific role in the body. The process by which this second cell becomes a specific type of cell is called differentiation. The body contains many different types of stem cells, such as neural stem cells, which go on to form the nervous system, and epithelial stem cells, which give rise to various types of surfaces in the body, such as the skin and the lining of the intestine.
Many types of epithelial cells are polarized, which means they have three distinct sides or domains: a basal domain that faces the underlying tissue; an apical domain on the opposite side; and a lateral domain on the side in between the apical and basal domains. The details of how cell polarity is established in epithelial cells are not fully understood, but it is thought to have its origins in the division of epithelial stem cells.
Now, by studying follicle stem cells in the ovaries of fruit flies, Castanieto et al. have shown that a process called EGFR signaling (which is short for epidermal growth factor receptor signaling) has a central role in establishing the difference between the stem cell and the cell that differentiates. EGFR signaling does this, in part, by promoting a ‘partially polarized state’ in the stem cells: this state is characterized by the presence of a basal domain and a lateral domain but no apical domain.
In fully polarized cells, the apical and lateral domains work together to ensure that all three domains remain separated on the surface of the cell, so it was surprising to find that the stem cell could maintain basal and lateral domains without an apical domain. Castanieto et al. propose that this feat is achieved by EGFR signaling, which activates a multiple number of proteins, including one called LKB1 that is known to regulate cell polarity.
This work strongly suggests that that changes in cell polarity are among the earliest differences to arise between epithelial stem cells and differentiating cells. In the future, it will be important to determine whether these differences in cell polarity cause the stem cells and the differentiating cells to take on different roles in the tissue. For example, it may be that the lack of an apical domain in the stem cells shields them from signals in the tissue that promote differentiation, thus allowing them to remain undifferentiated. Conversely, the development of an apical domain in the differentiating cells may expose them to signals that promote their differentiation, and also allow them to form a barrier and perform the other roles of epithelial tissue.
PMCID: PMC4298699  PMID: 25437306
cell polarity; EGFR; niche; epithelial cells; D. melanogaster
15.  Inhibition of SIRT1 Reactivates Silenced Cancer Genes without Loss of Promoter DNA Hypermethylation 
PLoS Genetics  2006;2(3):e40.
The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5′ CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)–mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.
The propensity for cancer to arise and progress is influenced not only by gene mutations (genetic abnormalities), but also by defects in gene expression programs that are inherited from one dividing cell to another. This change in the inheritance of gene expression patterns not associated with changes in the primary DNA sequence is referred to as an epigenetic abnormality. In virtually every form of cancer, tumor suppressor genes (TSGs) and candidate TSGs are epigenetically altered such that the ability of these genes to become activated and lead to production of the corresponding proteins is lost. This so-called gene “silencing” is often linked with abnormal accumulation of methyl groups to DNA (DNA methylation) in a region of the gene that controls its expression. The SIRT1 protein is an enzyme that can remove acetyl groups attached to specific amino acids in a number of different protein targets and thereby regulate gene silencing in yeast. However, in mammalian cells this has not been demonstrated. Here, the authors show SIRT1 is involved in epigenetic silencing of DNA-hypermethylated TSGs in cancer cells. Inhibition of SIRT1 by multiple approaches leads to TSG re-expression and a block in tumor-causing networks of cell signaling that are activated by loss of the TSGs in a wide range of cancers. This finding has important ramifications for the biology of cancer in terms of what maintains abnormal gene silencing. Furthermore, the authors propose that their observations may have potential clinical relevance in suggesting new means for restoring expression of abnormally silenced genes in cancer.
PMCID: PMC1420676  PMID: 16596166
16.  Transgenerational Propagation and Quantitative Maintenance of Paternal Centromeres Depends on Cid/Cenp-A Presence in Drosophila Sperm 
PLoS Biology  2012;10(12):e1001434.
Analysis of centromeres in progeny of Drosophila sperm with experimentally altered centromere-specific histone CenH3 levels reveals quantitative inheritance of this epigenetic mark.
In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.
Author Summary
Genetic information in eukaryotic cells is parceled into chromosomes. These information strings are precisely transmitted to daughter cells during mitotic and meiotic cell divisions, but only if the centromere, a specialized chromosomal region, is functional. The centromere region within chromosomes of many species—including humans and the fly Drosophila melanogaster—is thought to be specified epigenetically by incorporation of a centromere-specific histone H3 variant (CenH3). After chromosome replication, the centromeres in the resulting two sister chromatids might be expected to be composed of a mixture of pre-existing CenH3 evenly distributed onto the two copies during replication and new CenH3 recruited by the partitioned pool in a stoichiometric manner. Here, we have addressed whether centromeres are indeed replicated in this manner by experimentally altering the levels of centromeric CenH3 in Drosophila sperm. We show that centromeres on paternal chromosomes cannot recruit new CenH3 in embryos fertilized with sperm lacking CenH3. By using sperm with increased or reduced amounts of centromeric CenH3, we demonstrate that altered CenH3 levels are at least partially propagated on paternal centromeres throughout development of the offspring. We conclude that pre-existing CenH3 in Drosophila sperm is therefore not only required for transgenerational centromere maintenance, but that it also exerts quantitative control of this process.
PMCID: PMC3531477  PMID: 23300376
17.  Evidence of Activity-Specific, Radial Organization of Mitotic Chromosomes in Drosophila 
PLoS Biology  2011;9(1):e1000574.
A fluorescently labeled protein specifically binding to genes was reproducibly found at the periphery of condensed mitotic fruit fly chromosomes, illustrating preservation of a radial structure between consecutive divisions.
The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.
Author Summary
Mitotic chromosomes of eukaryotes are relatively large rod-like cellular organelles, about 1 µm in diameter and 10 µm long, of well-studied composition but unknown structure. The question of whether all DNA sequences equally contribute to the interactions leading to the formation of mitotic chromosomes has never been asked. To find an answer, we determined whether the radial positions of specific chromatin loci within mitotic chromosomes were reproduced at every cell cycle or were purely random. Based on fluorescence microscopy images of live or fixed chromosomes in cells from Drosophila embryos or Drosophila larval tissues expressing the MSL3-GFP fusion protein from a transgene, we report that the large-scale organization of mitotic chromosomes is reproduced not only longitudinally, as in the well-known chromosome banding phenomenon, but also radially. Actively transcribed, dosage-compensated genes of the Drosophila male X chromosome were always found at the periphery of mitotic chromosomes, starting from late prophase. Histone modifications specific to active chromatin were found to be more peripheral compared to silent chromatin that tended to be more central in the condensed chromosome. These findings are both exciting and significant for the field of cell and chromatin biology because they may help reconcile the old controversy between the existing models of chromosome structure that posit either radial loops of chromatin or consecutive coiling. In addition, we offer new insights into the mechanisms of mitotic condensation and suggest a link between structural and functional roles of different chromatin domains.
PMCID: PMC3019107  PMID: 21264350
18.  Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster 
PLoS ONE  2011;6(9):e25087.
Production of specialized cells from precursors depends on a tightly regulated sequence of proliferation and differentiation steps. In the gonad of Drosophila melanogaster, the daughters of germ line stem cells (GSC) go through precisely four rounds of transit amplification divisions to produce clusters of 16 interconnected germ line cells before entering a stereotypic differentiation cascade. Here we show that animals harbouring a transposon insertion in the center of the complex nucleoporin98-96 (nup98-96) locus had severe defects in the early steps of this developmental program, ultimately leading to germ cell loss and sterility. A phenotypic analysis indicated that flies carrying the transposon insertion, designated nup98-962288, had dramatically reduced numbers of germ line cells. In contrast to controls, mutant testes contained many solitary germ line cells that had committed to differentiation as well as abnormally small clusters of two, four or eight differentiating germ line cells. This indicates that mutant GSCs rather differentiated than self-renewed, and that these GSCs and their daughters initiated the differentiation cascade after zero, or less than four rounds of amplification divisions. This phenotype remained unaffected by hyper-activation of signalling pathways that normally result in excessive proliferation of GSCs and their daughters. Expression of wildtype nup98-96 specifically in the germ line cells of mutant animals fully restored development of the GSC lineage, demonstrating that the effect of the mutation is cell-autonomous. Nucleoporins are the structural components of the nucleopore and have also been implicated in transcriptional regulation of specific target genes. The nuclear envelopes of germ cells and general nucleocytoplasmic transport in nup98-96 mutant animals appeared normal, leading us to propose that Drosophila nup98-96 mediates the transport or transcription of targets required for the developmental timing between amplification and differentiation.
PMCID: PMC3174998  PMID: 21949861
19.  Role of DLC1 tumor suppressor gene and MYC oncogene in pathogenesis of human hepatocellular carcinoma: Potential prospects for combined targeted therapeutics 
International Journal of Oncology  2012;41(2):393-406.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, and its incidence is increasing worldwide in an alarming manner. The development of curative therapy for advanced and metastatic HCC is a high clinical priority. The HCC genome is complex and heterogeneous; therefore, the identification of recurrent genomic and related gene alterations is critical for developing clinical applications for diagnosis, prognosis and targeted therapy of the disease. This article focuses on recent research progress and our contribution in identifying and deciphering the role of defined genetic alterations in the pathogenesis of HCC. A significant number of genes that promote or suppress HCC cell growth have been identified at the sites of genomic reorganization. Notwithstanding the accumulation of multiple genetic alterations, highly recurrent changes on a single chromosome can alter the expression of oncogenes and tumor suppressor genes (TSGs) whose deregulation may be sufficient to drive the progression of normal hepatocytes to malignancy. A distinct and highly recurrent pattern of genomic imbalances in HCC includes the loss of DNA copy number (associated with loss of heterozygosity) of TSG-containing chromosome 8p and gain of DNA copy number or regional amplification of protooncogenes on chromosome 8q. Even though 8p is relatively small, it carries an unusually large number of TSGs, while, on the other side, several oncogenes are dispersed along 8q. Compelling evidence demonstrates that DLC1, a potent TSG on 8p, and MYC oncogene on 8q play a critical role in the pathogenesis of human HCC. Direct evidence for their role in the genesis of HCC has been obtained in a mosaic mouse model. Knockdown of DLC1 helps MYC in the induction of hepatoblast transformation in vitro, and in the development of HCC in vivo. Therapeutic interventions, which would simultaneously target signaling pathways governing both DLC1 and MYC functions in hepatocarcinogenesis, could result in progress in the treatment of liver cancer.
PMCID: PMC3583004  PMID: 22580498
DLC1; tumor suppressor; MYC; oncogene; liver cancer; targeted therapy
20.  Mesenchymal Stem Cells Modulate Albumin-Induced Renal Tubular Inflammation and Fibrosis 
PLoS ONE  2014;9(3):e90883.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) have recently shown promise as a therapeutic tool in various types of chronic kidney disease (CKD) models. However, the mechanism of action is incompletely understood. As renal prognosis in CKD is largely determined by the degree of renal tubular injury that correlates with residual proteinuria, we hypothesized that BM-MSCs may exert modulatory effects on renal tubular inflammation and epithelial-to-mesenchymal transition (EMT) under a protein-overloaded milieu. Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, we showed that concomitant stimulation of BM-MSCs by albumin excess was a prerequisite for them to attenuate albumin-induced IL-6, IL-8, TNF-α, CCL-2, CCL-5 overexpression in PTECs, which was partly mediated via deactivation of tubular NF-κB signaling. In addition, albumin induced tubular EMT, as shown by E-cadherin loss and α-SMA, FN and collagen IV overexpression, was also prevented by BM-MSC co-culture. Albumin-overloaded BM-MSCs per se retained their tri-lineage differentiation capacity and overexpressed hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 via P38 and NF-κB signaling. Albumin-induced tubular CCL-2, CCL-5 and TNF-α overexpression were suppressed by recombinant HGF treatment, while the upregulation of α-SMA, FN and collagen IV was attenuated by recombinant TSG-6. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively. In vivo, albumin-overloaded mice treated with mouse BM-MSCs had markedly reduced BUN, tubular CCL-2 and CCL-5 expression, α-SMA and collagen IV accumulation independent of changes in proteinuria. These data suggest anti-inflammatory and anti-fibrotic roles of BM-MSCs on renal tubular cells under a protein overloaded condition, probably mediated via the paracrine action of HGF and TSG-6.
PMCID: PMC3960109  PMID: 24646687
21.  Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage 
Nature  2008;454(7201):241-245.
Drosophila neuroblasts1 and ovarian stem cells2,3 are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim–NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells4-6, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim–NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.
PMCID: PMC2988194  PMID: 18528333
22.  The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells 
The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.
PMCID: PMC3923918  PMID: 24084155
Developmental Biology; Issue 79; Eye; Photoreceptor Cells; Genes; Developmental; neuron; visualization; degeneration; development; live imaging; Drosophila; photoreceptor; cornea neutralization; mitotic recombination
23.  Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast 
PLoS Biology  2009;7(10):e1000221.
The asymmetric localization of cell fate determinants results in asymmetric cell cycle control in budding yeast.
In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.
Author Summary
Asymmetric cell division is a universal mechanism for generating differentiated cells. The progeny of such divisions can often display differential cell cycle regulation. This study addresses how differential regulation of gene expression in the progeny of a single division can alter cell cycle control. In budding yeast, asymmetric cell division yields a bigger ‘mother’ cell and a smaller ‘daughter’ cell. Regulation of gene expression is also asymmetric because two transcription factors, Ace2 and Ash1, are specifically localized to the daughter. Cell size has long been proposed as important for the regulation of the cell cycle in yeast. Our work shows that Ace2 and Ash1 regulate size control in daughter cells: daughters ‘interpret’ their size as smaller, making size control more stringent and delaying cell cycle commitment relative to mother cells of the same size. This asymmetric interpretation of cell size is associated with differential regulation of the G1 cyclin CLN3 by Ace2 and Ash1, at least in part via direct binding of these factors to the CLN3 promoter. CLN3 is the most upstream regulator of Start, the initiation point of the yeast cell cycle, and differential regulation of CLN3 accounts for most or all asymmetric regulation of Start in budding yeast mother and daughter cells.
PMCID: PMC2756959  PMID: 19841732
24.  Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division 
eLife  2014;3:e02875.
Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.
eLife digest
A stem cell can divide in two ways. Either it can split symmetrically into two identical daughter stem cells, or it can split asymmetrically into a stem cell and a specialist cell. The structure that forms inside the dividing cell to separate pairs of chromosomes—called the mitotic spindle—also partitions the molecules that determine what kind of cell each daughter cell will become.
The mitotic spindle is made up of protein microtubules. Astral microtubules connect the spindle to a structure found at the inner face of the cell membrane called the cell cortex. This helps the spindle to orient itself correctly and control the plane of cell division. This is particularly important in cells that are different at their top and bottom, like polarized neural stem cells.
To divide symmetrically, these cells need to split vertically from top to bottom. Then, to divide asymmetrically they tilt the cell division plane off-vertical. Classical studies on neuroblasts from the fruit fly Drosophila have shown that a big, 90° reorientation, from vertical to horizontal underlies this change. However, in the primary stem cells of the mammalian brain, subtle off-vertical tilting suffices for asymmetric divisions to occur. This tilting must be finely regulated: if not, neurodevelopmental disorders, such as microcephaly and lissencephaly, may arise.
Mora-Bermúdez et al. investigated how mammalian cortical stem cells control such subtle spindle orientation changes by taking images of developing brain tissue from genetically modified mice. These show that not all astral microtubules affect whether the spindle reorients, as was previously thought. Instead, only those connecting the spindle to the cell cortex at the top and bottom of the cell—the apical/basal astrals—are involved.
A decrease in the number of apical/basal astrals enables the spindle to undergo small reorientations. Mora-Bermúdez et al. therefore propose a model in which the spindle becomes less strongly anchored when the number of apical/basal astrals is reduced. This makes the spindle easier to tilt, allowing neural stem cells to undergo asymmetric divisions to produce neurons.
The decrease in the number of apical/basal astrals appears to be caused by a reduction in the amount of a molecule that is known to help link the microtubules to the cell cortex. This reduction occurs only in the cortex at the top of the cell. Mora-Bermúdez et al. were also able to manipulate this process by adding very low doses of a microtubule inhibitor called nocodazole, which reduced the number of only the apical/basal astrals, increasing the ability of the spindle to reorient.
PMCID: PMC4112548  PMID: 24996848
asymmetric cell division; spindle orientation; neural stem cells; astral microtubules; neurogenesis; nocodazole; mouse
25.  Intra-lineage Directional Notch Signaling Regulates Self-renewal and Differentiation of Asymmetrically Dividing Radial Glia 
Neuron  2012;74(1):65-78.
Asymmetric division of progenitor/stem cells generates both self-renewing and differentiating progeny and is fundamental to development and regeneration. How this process is regulated in the vertebrate brain remains incompletely understood. Here we use time-lapse imaging to track radial glia progenitor behavior in the developing zebrafish brain. We find that asymmetric division invariably generates a basal self-renewing daughter and an apical differentiating sibling. Gene expression and genetic mosaic analysis further show that the apical daughter is the source of Notch ligand that is essential to maintain higher Notch activity in the basal daughter. Notably, establishment of this intra-lineage and directional Notch signaling requires the intrinsic polarity regulator Partitioning defective protein-3 (Par-3), which segregates the fate determinant Mind bomb unequally to the apical daughter, thereby restricting the self-renewal potential to the basal daughter. These findings reveal with single-cell resolution how self-renewal and differentiation become precisely segregated within asymmetrically dividing neural progenitor/stem lineages.
PMCID: PMC3466114  PMID: 22500631
single cell imaging analysis in vivo; proliferation; cancer; dysplasia; neural stem cell; clonal analysis; in vivo lineage tracing; interkinetic nuclear migration (INM)

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