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In JoVE (1)
Other Publications (8)
Articles by Douglas R. Mackay in JoVE
JoVE General
Time-lapse Imaging of Mitosis After siRNA Transfection
1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, 2Fluorescence Microscopy Core Facility, University of Utah
Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.
Other articles by Douglas R. Mackay on PubMed
The LEF1/beta -catenin Complex Activates Movo1, a Mouse Homolog of Drosophila Ovo Required for Epidermal Appendage Differentiation
Drosophila ovo/svb (dovo) is required for epidermal cuticle/denticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of dovo, movo1, is required for the proper formation of hair, a mammalian epidermal appendage. Here, we provide biochemical evidence that movo1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those that dOvo binds to, further supporting the notion that mOvo1a and dOvo are genetically and biochemically homologous proteins. Additionally, we show that the movo1 promoter is activated by the lymphoid enhancer factor 1 (LEF1)/beta-catenin complex, a transducer of wnt signaling. Collectively, our findings suggest that movo1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg/wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies.
Ovol2, a Mammalian Homolog of Drosophila Ovo: Gene Structure, Chromosomal Mapping, and Aberrant Expression in Blind-sterile Mice
The ovo gene family consists of evolutionarily conserved genes including those cloned from Caenorhabditis elegans, Drosophila melanogaster, mouse, and human. Here we report the isolation and characterization of mouse Ovol2 (also known as movol2 or movo2) and provide evidence supporting the existence of multiple Ovol2 transcripts. These transcripts are produced by alternative promoter usage and alternative splicing and encode long and short OVOL2 protein isoforms, whose sequences differ from those previously reported. Mouse and human OVOL2 genes are expressed in overlapping tissues including testis, where Ovol2 expression is developmentally regulated and correlates with the meiotic/postmeiotic stages of spermatogenesis. Mouse Ovol2 maps to chromosome 2 in a region containing blind-sterile (bs), a spontaneous mutation that causes spermatogenic defects and germ cell loss. No mutation has been detected in the coding region of Ovol2 from bs mice, but Ovol2 transcription was dramatically reduced in testes from these mice, suggesting that Ovol2 is expressed in male germ cells.
Cloning and Developmental Expression of Mouse Pygopus 2, a Putative Wnt Signaling Component
Recent studies in Drosophila identified pygopus, which encodes a PHD finger protein, as an additional nuclear component of the canonical Wingless(Wg)/Wnt signaling pathway. In this study, we describe the molecular cloning and expression analysis of a mouse pygopus gene, mpygo2. mpygo2 transcripts were detected in almost all adult mouse tissues examined, whereas transcripts of another mouse pygopus gene, mpygo1, were detected only in heart tissue. Abundant mpygo2 transcripts were observed during embryogenesis in multiple developmental sites. Consistent with the demonstrated role of the Wnt-beta-catenin-LEF/TCF signaling pathway in mammalian skin development, mpygo2 expression was detected in the developing epidermis and hair follicles, which suggests that mpygo2 might mediate the effect of this signaling pathway in mouse skin.
Ovol1 Regulates Meiotic Pachytene Progression During Spermatogenesis by Repressing Id2 Expression
Previous studies have shown that a targeted deletion of Ovol1 (previously known as movo1), encoding a member of the Ovo family of zinc-finger transcription factors, leads to germ cell degeneration and defective sperm production in adult mice. To explore the cellular and molecular mechanism of Ovol1 function, we have examined the mutant testis phenotype during the first wave of spermatogenesis in juvenile mice. Consistent with the detection of Ovol1 transcripts in pachytene spermatocytes of the meiotic prophase, Ovol1-deficient germ cells were defective in progressing through the pachytene stage. The pachytene arrest was accompanied by an inefficient exit from proliferation, increased apoptosis and an abnormal nuclear localization of the G2-M cell cycle regulator cyclin B1, but was not associated with apparent chromosomal or recombination defects. Transcriptional profiling and northern blot analysis revealed reduced expression of pachytene markers in the mutant, providing molecular evidence that pachytene differentiation was defective. In addition, the expression of Id2 (inhibitor of differentiation 2), a known regulator of spermatogenesis, was upregulated in Ovol1-deficient pachytene spermatocytes and repressed by Ovol1 in reporter assays. Taken together, our studies demonstrate a role for Ovol1 in regulating pachytene progression of male germ cells, and identify Id2 as a Ovol1 target.
The Mouse Ovol2 Gene is Required for Cranial Neural Tube Development
The Ovo gene family encodes a group of evolutionarily conserved transcription factors and includes members that reside downstream of key developmental signaling pathways such as Wg/Wnt and BMP/TGF-beta. In the current study, we explore the function of Ovol2, one of three Ovo paralogues in mice. We report that Ovol2 is expressed during early-mid embryogenesis, particularly in the inner cell mass at E3.5, in epiblast at E6.5, and at later stages in ectodermally derived tissues such as the rostral surface (epidermal) ectoderm. Embryos in which Ovol2 is ablated exhibit lethality by E10.5, prior to which they display severe defects including an open cranial neural tube. The neural defects are associated with improper Shh expression in the underlying rostral axial mesoderm and localized changes of neural marker expression along the dorsoventral axis, as well as with expanded cranial neural tissue and reduced cranial surface ectoderm culminating in a lateral shift of the neuroectoderm/surface ectoderm border. We propose that these defects reflect the involvement of Ovol2 in independent processes such as regionalized gene expression and neural/non-neural ectodermal patterning. Additionally, we present evidence that Ovol2 is required for efficient migration and survival of neural crest cells that arise at the neuroectoderm/surface ectoderm border, but not for their initial formation. Collectively, our studies indicate that Ovol2 is a key regulator of neural development and reveal a previously unexplored role for Ovo genes in mammalian embryogenesis.
The Nucleoporin Nup153 Has Separable Roles in Both Early Mitotic Progression and the Resolution of Mitosis
Accurate inheritance of genomic content during cell division is dependent on synchronized changes in cellular organization and chromosome dynamics. Elucidating how these events are coordinated is necessary for a complete understanding of cell proliferation. Previous in vitro studies have suggested that the nuclear pore protein Nup153 is a good candidate for participating in mitotic coordination. To decipher whether this is the case in mammalian somatic cells, we reduced the levels of Nup153 in HeLa cells and monitored consequences on cell growth. Reduction of Nup153 resulted in a delay during the late stages of mitosis accompanied by an increase in unresolved midbodies. Depletion of Nup153 to an even lower threshold led to a pronounced defect early in mitosis and an accumulation of cells with multilobed nuclei. Although global nucleocytoplasmic transport was not significantly altered under these depletion conditions, the FG-rich region of Nup153 was required to rescue defects in late mitosis. Thus, this motif may play a specialized role as cells exit mitosis. Rescue of the multilobed nuclei phenotype, in contrast, was independent of the FG-domain, revealing two separable roles for Nup153 in the execution of mitosis.
Defects in Nuclear Pore Assembly Lead to Activation of an Aurora B-mediated Abscission Checkpoint
Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (Nup 153) is required for timely progression through late mitosis. In this study, we report that disruption of Nup 153 function by either small interfering RNA-mediated depletion or expression of a dominant-interfering Nup 153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup 50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B-dependent delay in abscission. Depletion of Nup 50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B-mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present.
Coordinating Postmitotic Nuclear Pore Complex Assembly with Abscission Timing
Cells divide and accurately inherit genomic and cellular content through synchronized changes in cellular organization and chromosome dynamics. Although DNA segregation, nuclear reformation, and cytokinesis/abscission temporally overlap, little is known about how these distinct events are coordinated to ensure accurate cell division. Recently, we found that disruption of postmitotic nuclear pore complex assembly, an essential aspect of the newly forming nuclear envelope, triggers an Aurora B-dependent delay in abscission. This delay is further characterized by mislocalized, aberrantly active Aurora B in the cytoplasm of midbody-stage cells. These results support a model in which an Aurora B-mediated abscission checkpoint provides surveillance of nuclear pore complex formation to ensure that elements of nuclear architecture are fully formed before daughter cells are physically separated. Here we discuss the process of nuclear pore complex assembly, describe potential mechanisms that may explain how this process could be coordinated with abscission, and postulate why such a checkpoint mechanism may exist.
