Live imaging of Drosophila larval neuroblasts.

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

[1]  M. Peifer,et al.  A role for a novel centrosome cycle in asymmetric cell division , 2007, The Journal of cell biology.

[2]  J. Reina,et al.  Centrobin controls mother–daughter centriole asymmetry in Drosophila neuroblasts , 2013, Nature Cell Biology.

[3]  C. Doe Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system. , 1992, Development.

[4]  Juergen A. Knoblich,et al.  Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages , 2013, PloS one.

[5]  H. Reichert,et al.  Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development , 2008, Neural Development.

[6]  C. Rieder,et al.  Flattening Drosophila cells for high-resolution light microscopic studies of mitosis in vitro. , 2003, Cell motility and the cytoskeleton.

[7]  C. Doe,et al.  Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts , 1997, Current Biology.

[8]  N. Rusan,et al.  PLP inhibits the activity of interphase centrosomes to ensure their proper segregation in stem cells , 2013, The Journal of cell biology.

[9]  C. Doe,et al.  Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts , 2006, Development.

[10]  M. Peifer,et al.  Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting? , 2008, The Journal of cell biology.

[11]  Chris Q Doe,et al.  Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells , 2008, Developmental neurobiology.

[12]  R. Steward,et al.  Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control. , 2005, Molecular biology of the cell.

[13]  J. Raff,et al.  Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila Centrosomin , 2007, The Journal of cell biology.

[14]  Juergen A. Knoblich,et al.  Genome-Wide Analysis of Self-Renewal in Drosophila Neural Stem Cells by Transgenic RNAi , 2011, Cell stem cell.

[15]  C. Sunkel,et al.  In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila. , 1999, Biology of the cell.

[16]  J. Raff,et al.  Cnn Dynamics Drive Centrosome Size Asymmetry to Ensure Daughter Centriole Retention in Drosophila Neuroblasts , 2010, Current Biology.

[17]  M. Bate,et al.  Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. , 1988, Developmental biology.

[18]  D. Montell,et al.  A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging , 2007, Nature Protocols.

[19]  J. Knoblich,et al.  Drosophila neuroblasts: a model for stem cell biology , 2012, Development.

[20]  M. Martinez-Campos,et al.  The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis , 2004, The Journal of cell biology.

[21]  C. Doe,et al.  Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila. , 2009, Developmental cell.

[22]  S. Bowman,et al.  The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila. , 2008, Developmental cell.

[23]  R. Karess,et al.  Recruitment of Mad2 to the Kinetochore Requires the Rod/Zw10 Complex , 2005, Current Biology.

[24]  A. Spradling,et al.  Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary , 2011, Development.

[25]  N. Rusan,et al.  Organelle asymmetry for proper fitness, function, and fate , 2013, Chromosome Research.

[26]  J. Pickett-Heaps,et al.  Cytochalasin D and Latrunculin Affect Chromosome Behaviour During Meiosis in Crane-fly Spermatocytes , 1998, Chromosome Research.

[27]  J. Raff,et al.  Drosophila Spd-2 Recruits PCM to the Sperm Centriole, but Is Dispensable for Centriole Duplication , 2007, Current Biology.

[28]  J. S. Britton,et al.  Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. , 1998, Development.

[29]  J. Reina,et al.  Drosophila neuroblasts retain the daughter centrosome , 2011, Nature communications.

[30]  C. Gonzalez,et al.  The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts , 2010, The Journal of cell biology.

[31]  F. Turner,et al.  The centrosome is a dynamic structure that ejects PCM flares , 2002, Journal of Cell Science.

[32]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[33]  C. Gonzalez,et al.  Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. , 2007, Developmental cell.

[34]  C. Doe,et al.  Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts. , 2008, Developmental biology.

[35]  Chris Q Doe,et al.  Live imaging of neuroblast lineages within intact larval brains in Drosophila. , 2013, Cold Spring Harbor protocols.

[36]  J. Raff,et al.  Flies without Centrioles , 2006, Cell.

[37]  C. Rieder,et al.  Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts. , 2002, Journal of cell science.

[38]  Alexey Khodjakov,et al.  Centrosome Amplification Can Initiate Tumorigenesis in Flies , 2008, Cell.