Mosaic Analysis With A Repressible Cell Marker Research Articles

Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system

Most neurons of the adult Drosophila ventral nerve cord arise from a burst of neurogenesis during the third larval instar stage. Most of this growth occurs in thoracic neuromeres, which contain 25 individually identifiable postembryonic neuronal lineages. Initially, each lineage consists of two hemilineages--'A' (Notch(On)) and 'B' (Notch(Off))--that exhibit distinct axonal trajectories or fates. No reliable method presently exists to identify these lineages or hemilineages unambiguously other than labor-intensive lineage-tracing methods. By combining mosaic analysis with a repressible cell marker (MARCM) analysis with gene expression studies, we constructed a gene expression map that enables the rapid, unambiguous identification of 23 of the 25 postembryonic lineages based on the expression of 15 transcription factors. Pilot genetic studies reveal that these transcription factors regulate the specification and differentiation of postembryonic neurons: for example, Nkx6 is necessary and sufficient to direct axonal pathway selection in lineage 3. The gene expression map thus provides a descriptive foundation for the genetic and molecular dissection of adult-specific neurogenesis and identifies many transcription factors that are likely to regulate the development and differentiation of discrete subsets of postembryonic neurons.

GABAergic circuit dysfunction in the Drosophila Fragile X syndrome model

Fragile X syndrome (FXS), caused by loss of FMR1 gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 protein (FMRP) translational regulator mediates activity-dependent control of synapses. In addition to the metabotropic glutamate receptor (mGluR) hyperexcitation FXS theory, the GABA theory postulates that hypoinhibition is causative for disease state symptoms. Here, we use the Drosophila FXS model to assay central brain GABAergic circuitry, especially within the Mushroom Body (MB) learning center. All 3 GABAA receptor (GABAAR) subunits are reportedly downregulated in dfmr1 null brains. We demonstrate parallel downregulation of glutamic acid decarboxylase (GAD), the rate-limiting GABA synthesis enzyme, although GABAergic cell numbers appear unaffected. Mosaic analysis with a repressible cell marker (MARCM) single-cell clonal studies show that dfmr1 null GABAergic neurons innervating the MB calyx display altered architectural development, with early underdevelopment followed by later overelaboration. In addition, a new class of extra-calyx terminating GABAergic neurons is shown to include MB intrinsic α/β Kenyon Cells (KCs), revealing a novel level of MB inhibitory regulation. Functionally, dfmr1 null GABAergic neurons exhibit elevated calcium signaling and altered kinetics in response to acute depolarization. To test the role of these GABAergic changes, we attempted to pharmacologically restore GABAergic signaling and assay effects on the compromised MB-dependent olfactory learning in dfmr1 mutants, but found no improvement. Our results show that GABAergic circuit structure and function are impaired in the FXS disease state, but that correction of hypoinhibition alone is not sufficient to rescue a behavioral learning impairment.

The Drosophila Importin-α3 Is Required for Nuclear Import of Notch In Vivo and It Displays Synergistic Effects with Notch Receptor on Cell Proliferation

The Notch signaling pathway controls diverse cell-fate specification events throughout development. The versatility of this pathway to influence different aspects of development comes from its multiple levels of regulation. Upon ligand-induced Notch activation, the Notch intracellular domain (Notch-ICD) is released from the membrane and translocates to the nucleus, where it transduces Notch signals by regulating the transcription of downstream target genes. But the exact mechanism of translocation of Notch-ICD into the nucleus is not clear. Here, we implicate Importin-α3 (also known as karyopherin-α3) in the nuclear translocation of Notch-ICD in Drosophila. Our present analyses reveal that Importin-α3 can directly bind to Notch-ICD and loss of Importin-α3 function results in cytoplasmic accumulation of the Notch receptor. Using MARCM (Mosaic Analysis with a Repressible Cell Marker) technique, we demonstrate that Importin-α3 is required for nuclear localization of Notch-ICD. These results reveal that the nuclear transport of Notch-ICD is mediated by the canonical Importin-α3/Importin-β transport pathway. In addition, co-expression of both Notch-ICD and Importin-α3 displays synergistic effects on cell proliferation. Taken together, our results suggest that Importin-α3 mediated nuclear import of Notch-ICD may play important role in regulation of Notch signaling.

Clonal Analysis of Olfaction in Drosophila: Immunochemistry and Imaging of Fly Brains

Clonal analysis with the MARCM (mosaic analysis with a repressible cell marker) system can be used for studying cell lineage, development, and anatomy in the Drosophila olfactory system and other parts of the fly brain. This protocol describes the dissection, staining, and imaging of brains from Drosophila with mosaic labeling. Staining for the presynaptic marker Bruchpilot (nc82) is performed in the example given here. The well-stained whole brain images that are obtained can be used to examine neuronal morphology. They are of sufficient quality to be used for image registration, which allows one to compare confocal images of labeled neurons in different brains.

Clonal analysis of olfaction in Drosophila: image registration.

Clonal analysis with the MARCM (mosaic analysis with a repressible cell marker) system can be used for studying cell lineage, development, and anatomy in the Drosophila olfactory system and other parts of the fly brain. To compare confocal images of labeled neurons in different brains, it may be desirable to register them to a template or standard brain. There are various image registration approaches available. Some depend on manually specifying landmarks on the brains to be registered. Others depend only on the grayscale intensity value of one of the channels in the confocal image. Another important difference between registration approaches is whether they apply linear or nonlinear (warping) transformations. Linear transformations typically include translation, rotation, and scaling along each axis. Nonlinear transformations are much more computationally intensive, but are required to register brains with different shapes. Here we describe the practical steps required for an intensity-based nonlinear registration that has been used to map the higher olfactory centers of the Drosophila brain using the staining for the presynaptic marker Bruchpilot (nc82). This registration is in fact a two-step process. The first step is a linear transformation that roughly aligns the two brains, followed by a second nonlinear step that allows different parts of the brain to move in slightly different directions.

Clonal analysis of olfaction in Drosophila: generation of flies with mosaic labeling.

Clonal analysis with the MARCM (mosaic analysis with a repressible cell marker) system can be used for studying cell lineage, development, and anatomy in the Drosophila olfactory system and other parts of the fly brain. This protocol gives a method for generating flies with mosaic labeling. It describes how to establish a mating cage for MARCM in PNs (projection neurons) of the fly antennal lobe and then select appropriate flies for dissection and staining using immunohistochemistry. The protocol can be adapted to determine the birth order of neuroblast lineages or individual cells. Alternatively, it can be used to dissect a complicated Gal4 line into its component neuroblast lineages to help elucidate projection patterns and connectivity. Collecting newly hatched larvae during a short time window allows for precise control of the stage during development at which the heat shock is applied.

Reverse Genetics by Loss-of-Function Mosaic Analysis in Drosophila

Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. One major application of MARCM, as described here, is to study cell-autonomous function(s) of a gene within single cells or a group of cells in otherwise unperturbed organisms. A mutation of interest distal to one FRT site is put in trans to a tubP-GAL80-containing chromosome arm that carries the same FRT. The resulting MARCM clones, which are negative for tubP-GAL80 and thus specifically marked, will become homozygous for the mutation in otherwise heterozygous organisms. By including a UAS-transgene, one can perform rescue experiments in the mutant MARCM clones. Conversely, if the mutation is placed on the same chromosome arm as tubP-GAL80, MARCM-labeled cells will be homozygous wild-type and may lie adjacent to sister cells that are homozygous mutant. This variant, called reverse MARCM, allows one to determine non-cell-autonomous effects of a mutation.

Genetic Mosaic Screens in Drosophila Mushroom Bodies

Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. In neurons whose progenitors can be efficiently targeted for mitotic recombination, genetic mosaic screens can be used to systematically uncover cell-autonomous genes that are required for development or function. This technique involves the generation of numerous FRT lines carrying various independent mutations, followed by derivation and phenotypic analysis of MARCM clones using these mutant FRT lines in combination with an MARCM-enabling stock that carries all the other genetic elements required for MARCM. Mutants of interest are recovered based on the MARCM phenotypes, which are imaged live using diverse fluorescent markers. Mutant genes that underlie the phenotypes of interest can then be identified by conventional genetics including derivation and analysis of series of recombinant chromosomes. Besides chemical mutagenesis, genes on a particular FRT chromosome may be randomly disrupted by P element insertion. This protocol describes procedures specifically used for genetic mosaic screens in the mushroom bodies (MBs).

Generation of Standard Wild-Type MARCM Clones for Analysis of Drosophila Brain Development

The generation and analysis of clones of cells with a genotype different from the rest of an organism has been used for studying molecular mechanisms underlying development. Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene. This permits unique labeling of specific homozygous daughter cells, and thus makes mosaic analysis possible in the complex nervous system. The principle of MARCM involves the generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues, therefore allowing GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, it is necessary to generate organisms carrying at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. Induction of FLP in neural precursors can be temporally controlled using a heat-shock promoter or spatially regulated using a tissue-specific promoter. Mitotic recombination in a neuroblast (NB) may yield a multicellular NB clone (progeny of the renewed NB) or a two-cell clone (progeny of the derived ganglion mother cell [GMC]). Single-cell clones can be obtained following mitotic recombination in GMCs. Phenotypic analysis of mosaic brains can be greatly simplified using a GAL4 driver that is only expressed in neurons of interest. One can also mark different subcellular structures of the clones using distinct UAS reporters. This protocol outlines the steps involved in standard MARCM analysis.

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

Progress on cell lineage analysis in <I>Drosophila melanogaster</I>

Lineage analysis of a single cell provides a powerful mean to delineate its functions during animal development, which, however, has been hindered by the complex nature of tissues that consist of many different types of cells with divergent morphologies, structures and functions. Mosaic technique and various labeling methods have provided ideal genetic tools for such studies. In this review, we described seven lineage analysis techniques that have been generally applied in Drosophila melanogaster, including FRT-mediated mitotic recombination, MARCM (Mosaic analysis with a repressible cell marker), TSG (Twin spot generator), Twin-spot MARCM, Q-MARCM (Q system-based MARCM), Coupled MARCM, and G-TRACE (Gal4 technique for real-time and clonal expression). These techniques enable researchers to perform genetic manipulations at a single cell level, and trace its development in complicated systems such as the nervous system. These methods may also be applied to lineage analysis in other model organisms.

Localized Netrins Act as Positional Cues to Control Layer-Specific Targeting of Photoreceptor Axons in Drosophila

SummaryA shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.

Programmed cell death in type II neuroblast lineages is required for central complex development in the Drosophila brain

BackgroundThe number of neurons generated by neural stem cells is dependent upon the regulation of cell proliferation and by programmed cell death. Recently, novel neural stem cells that amplify neural proliferation through intermediate neural progenitors, called type II neuroblasts, have been discovered, which are active during brain development in Drosophila. We investigated programmed cell death in the dorsomedial (DM) amplifying type II lineages that contribute neurons to the development of the central complex in Drosophila, using clonal mosaic analysis with a repressible cell marker (MARCM) and lineage-tracing techniques.ResultsA significant number of the adult-specific neurons generated in these DM lineages were eliminated by programmed cell death. Programmed cell death occurred during both larval and pupal stages. During larval development, approximately one-quarter of the neuronal (but not glial) cells in the lineages were eliminated by apoptosis before the formation of synaptic connectivity during pupal stages. Lineage-tracing experiments documented the extensive contribution of intermediate neural progenitor-containing DM lineages to all of the major modular substructures of the adult central complex. Moreover, blockage of apoptotic cell death specifically in these lineages led to prominent innervation defects of DM-derived neural progeny in the major neuropile substructures of the adult central complex.ConclusionsOur findings indicate that significant neural overproliferation occurs normally in type II DM lineage development, and that elimination of excess neurons in these lineages through programmed cell death is required for the formation of correct neuropile innervation in the developing central complex. Thus, amplification of neuronal proliferation through intermediate progenitors and reduction of neuronal number through programmed cell death operate in concert in type II neural stem-cell lineages during brain development.

Morphological Analysis of <em>Drosophila</em> Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation. There are four main DA neuron classes (I-IV)(1). They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation(2-10). The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology(11-13) because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses. DA neuron activity modifies the output of a larval locomotion central pattern generator(14-16). The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses(14,16-20). Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)(21). These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field(7,22,23). Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping(7,22,23), as well as the wiring of a simple circuit modulating larval locomotion(14-17). We present here a practical guide to generate and analyze genetic mosaics(24) marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)(1,10,25) and Flp-out(22,26,27) techniques (summarized in Fig. 1).

Morphological Analysis of <em>Drosophila</em> Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation. There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses. DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17. We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Generation and Staining of MARCM Clones in Drosophila: Figure 1.

INTRODUCTIONDendrite morphological diversity helps to define the properties of neural circuits by influencing circuit organization and information processing. Dendrite development has been shown to be driven by a combination of cell-intrinsic and -extrinsic factors. However, much remains to be discovered about the cellular and molecular mechanisms that give rise to unique yet highly stereotyped dendrite arbors of diverse neuronal types. Work in the last decade has established the Drosophila system as an excellent model for studies of dendrite morphogenesis. Mosaic analysis with a repressible cell marker (MARCM) permits resolution of dendrites at a single-cell level and genetic manipulation of individual neurons to assess gene function during neuronal morphogenesis. Mosaic systems for examining mutant phenotypes are advantageous for studies of larval stages because of the ability to discern cell-autonomous phenotypes at very high resolution. The protocol described here can be used to generate and label MARCM clones for the analysis of dendritic patterning and branching control in Drosophila.

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).

Sexually Dimorphic Shaping of Interneuron Dendrites Involves the Hunchback Transcription Factor

Sexual dimorphism of the brain has been well characterized anatomically in Drosophila melanogaster at the single neuron level, yet little is known about the molecular mechanism whereby cellular sex differences are generated except that the neural sex determination gene fruitless (fru) plays a key role. The fru-expressing mAL interneuron cluster is sexually dimorphic in three aspects: the number of cells composing the cluster is 5 in females and 30 in males; the ipsilateral neurite is absent in females and present in males; the contralateral neurite forms Y-shaped branches in the subesophageal ganglion in females while it ends with a simple horsetail-like structure in males. By screens in the compound eye for modifiers of roughness induced by fru(+) overexpression, we identified a loss-of-function allele of hunchback (hb) to be a suppressor of this phenotype. Hb was expressed in most of the fru-expressing neurons in the pupal and adult stages. Knocking down hb in mAL MARCM (Mosaic Analysis with a Repressible Cell Marker) clones in the male brain resulted in partial demasculinization of the branching pattern of the contralateral neurites without affecting the cell number and the ipsilateral neurite formation. The present results suggest that Hb is essential for male-typical shaping of the contralateral neurites by Fru.