A temporal mechanism that produces neuronal diversity in the Drosophila visual center

AbstractThe brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf), correspond to types of medulla neurons. Here, we examine the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, we searched for transcription factors that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates (Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)). The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. Our results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequentially expressed in NBs

Concentric zones, cell migration and neuronal circuits in the Drosophila visual center

金沢大学フロンティアサイエンス機構The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visual systems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla. Here, we describe the key features of the developing medulla and report novel phenomena that could accelerate our understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla.出版社許諾要件により、2012年3月より全文公開

Formation of Neuronal Circuits by Interactions between Neuronal Populations Derived from Different Origins in the Drosophila Visual Center

A wide variety of neurons, including populations derived from different origins, are precisely arranged and correctly connected with their partner to establish a functional neural circuit during brain development. The molecular mechanisms that orchestrate the production and arrangement of these neurons have been obscure. Here, we demonstrate that cell-cell interactions play an important role in establishing the arrangement of neurons of different origins in the Drosophila visual center. Specific types of neurons born outside the medulla primordium migrate tangentially into the developing medulla cortex. During their tangential migration, these neurons express the repellent ligand Slit, and the two layers that the neurons intercalate between express the receptors Robo2 and Robo3. Genetic analysis suggests that Slit-Robo signaling may control the positioning of the layer cells or their processes to form a path for migration. Our results suggest that conserved axon guidance signaling is involved in the interactions between neurons of different origins during brain development

The post-anaphase SUMO pathway ensures the maintenance of centromeric cohesion through meiosis I-II transition in mammalian oocytes

The production of haploid gametes requires the maintenance of centromeric cohesion between sister chromatids through the transition between two successive meiotic divisions, meiosis I and meiosis II. One mechanism for the cohesion maintenance is shugoshin-dependent protection of centromeric cohesin at anaphase I onset [1–3]. However, how centromeric cohesion is maintained during late anaphase I and telophase I, when centromeric shugoshin is undetectable [1–3], remains largely unexplored. Here we show that the centromeric small ubiquitin-related modifier (SUMO) pathway is critical for the maintenance of centromeric cohesion during post-anaphase-I periods in mouse oocytes. SUMO2/3 and E3 ligase PIAS are enriched near centromeres during late anaphase I and telophase I. Specific perturbation of the centromeric SUMO pathway results in precocious loss of centromeric cohesin at telophase I, although shugoshin-dependent centromeric protection at anaphase I onset remains largely intact. Prevention of the SUMO perturbation during post-anaphase-I periods restores the maintenance of centromeric cohesion through the meiosis I-II transition. Thus, the post-anaphase-I centromeric SUMO pathway ensures continuous maintenance of centromeric cohesion through the meiosis I-II transition.This work was supported by research grants JSPS KAKENHI26930008 (to M.K.), 24770173/16H01226/16H06161 (to T.S.K.), MINECO BFU2017-89408-R and CyLe (to A.M.P.), and RIKEN intramural grants.Peer Reviewe

Investigation of morphological changes for the discrimination of nucleated red blood cells and other leukocytes in Sysmex XN hematology analyzer scattergrams using transmission electron microscopy

Background: The WNR channel of the XN-Series automated hematology analyzer (Sysmex) counts white blood cells (WBCs) and simultaneously performs a differential counting of basophils and nucleated red blood cells (NRBCs). The detection process involves exposing the cells to WNR-specific reagents containing an acidic detergent and a fluorescent dye and measuring the intensity of the forward scattered light (FSC) and side fluorescence light (SFL). Method: We treated isolated peripheral WBCs and NRBCs with specific reagents and assessed the morphological changes in NRBCs and each leukocyte type using transmission electron microscopy (TEM). Results: The results from a flow cytometer (FCM) showed that, after exposure to the reagents, basophils appeared on the highest FSC and SFL areas compared to other leukocytes on the WNR scattergram. Owing to the hemolysis of reticulocytes and erythrocytes, NRBCs that survived the reagent treatment could be distinguished by their lower intensity than those of the other leukocytes on the WNR scattergram. We investigated the significance of the relationship between the TEM and FCM results after the reagent treatment. Conclusion: We confirmed that the WNR channel differentiates the blood cells on the WNR scattergram based on differences in the amount of residual cytoplasm and nucleic acids. Keywords: Automated hematology analyzer, Flow cytometry, Transmission electron microscopy, Leukocytes, NRBCs, Scatter light intensity, Fluorescent intensit

PLK1 activates the APC/C through multiple pathways.

<p>(A) Imaging of oocytes expressing securin-EGFP (green) and H2B-mCherry (chromosomes, red) in the presence of DMSO (control), 100 nM BI2536 and/or 1 μM reversine. Maximum intensity z-projection images are shown. Time after NEBD (h). Scale bar = 10 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s012" target="_blank">S5 Movie</a>. (B) Normalized intensities of cytoplasmic securin-EGFP signals (I_norm) were plotted. Average and s.d. are shown (n = 6, 4, 12, 15). (C-E) A line was fitted to the decrease of I_norm (C). Time for the start of securin-EGFP degradation (T_start) was defined as the time when the fitted line reaches the I_norm value 1.0. The degradation rate (D_rate) was defined as the negative value of the slope of the fitted line. Averages with s.d. of T_start and D_rate are shown in (D) and (E), respectively (n = 6, 12, 15. **p < 0.01).</p

PLK1 is required for chromosome alignment.

<p>(A) Imaging of meiosis I in oocytes expressing EGFP-CENP-C (kinetochores, green) and H2B-mCherry (chromosomes, red) in the presence of DMSO (control) or 100 nM BI2536. Maximum intensity z-projection images are shown. White lines indicate kinetochore tracks over 5 timepoints. Time after NEBD (h:mm). Scale bar = 10 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s011" target="_blank">S4 Movie</a>. (B) Kinetochore positions were determined from (A) and shown in the 3D plot as green spheres. Red bars connect homologous kinetochores. The view along the chromosome distribution equator (side view) is shown. Black arrowheads indicate misaligned chromosomes. Time after prometaphase belt formation (h:mm). The unit of the grid is 5 μm. (C) Chromosome positions along the estimated spindle axis were plotted for all twenty chromosomes of a single oocyte cultured in the presence of DMSO (control, black) or 100 nM BI2536 (red). (D) Distances between chromosomes and the equator at 0 and 2 hours after the prometaphase belt formation were potted. The box indicates 10–90 percentile (n = 60, 60, 60, 60 from three oocytes for each condition). ***p < 0.0001. (E) Oocytes 4 hours after NEBD were briefly treated with a cold buffer and fixed for immunostaining of microtubules (blue) and kinetochores (red). 100 nM BI2536 was added at 2 hours after NEBD. DNA was stained with Hoechst33342 (blue). Insets show magnified images of kinetochore-microtubule attachments. Scale bar = 10 μm. Average and s.d. of the population of unattached kinetochores are shown (n = 5, 5. **p < 0.01).</p

PLK1 is required for chromosome segregation, first polar body extrusion, and maintenance of the condensed state of chromosomes.

<p>(A) Experimental scheme. Oocytes were cultured for 6 hours in control medium, and then MG132 was added. The oocytes were incubated for 4 hours to arrest oocytes at the late metaphase I. After release from MG132, 100 nM BI2536 was added and oocytes were imaged. (B) Anaphase phenotypes after MG132 release in control and BI2536-treated oocytes. PB = the first polar body. (C) Imaging of securin-EGFP (green) and H2B-mCherry (red) after DMSO (control, top) or 100 nM BI2536 (lower panels) was added at the time of the MG132 washout. Each phenotype from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g006" target="_blank">Fig. 6B</a> is shown on a representative image sequence. Arrows indicate lagging chromosomes. Note that none of the BI2536-treated oocytes undergoing abnormal chromosome segregation extruded the first polar body. Time after MG132 washout (h:mm). Scale bar = 30 μm. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.s014" target="_blank">S7 Movie</a>. (D) Quantification of securin-EGFP destruction. Values were normalized to 1 when the imaging was started. Time relative to MG132 washout (h). The ‘BI2536 with anaphase’ curve represents BI2536-treated oocytes that underwent abnormal chromosome segregation either with or without DNA decondensation (3^rd and 4^th rows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g007" target="_blank">Fig. 7C</a>). The ‘BI2536 w/o anaphase’ curve represents BI2536-treated oocytes that did not undergo chromosome segregation (2^nd row in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116783#pone.0116783.g007" target="_blank">Fig. 7C</a>). Average and s.d. are shown (n = 23, 40). (E) Degradation rate of securin-EGFP calculated from (D). Average and s.d. are shown. ***p < 0.001.</p