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1.  Dynamic migration and cell-cell interactions of early reprogramming revealed by high resolution time-lapse imaging 
Stem cells (Dayton, Ohio)  2013;31(5):895-905.
Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multi-color time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as five minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct 2-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate the requirement of E-cadherin for proper cellular interactions from an early stage of reprogramming, including the 2-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.
doi:10.1002/stem.1323
PMCID: PMC4309553  PMID: 23335078
2.  Kinesin 5B (KIF5B) Is Required for Progression through Female Meiosis and Proper Chromosomal Segregation in Mitotic Cells 
PLoS ONE  2013;8(4):e58585.
The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.
doi:10.1371/journal.pone.0058585
PMCID: PMC3613343  PMID: 23560038
3.  Chimeric mice reveal clonal development of pancreatic acini, but not islets 
Intestinal crypt stem cells establish clonal descendants. To determine whether the pancreas is patterned by a similar process, we used embryonic stem (ES) cell chimeric mice, in which male ES cells were injected into female blastocysts. Fluorescence in situ hybridization for the Y chromosome (Y-FISH) revealed clonal patterning of ES-derived cells in the adult mouse small intestine and pancreas. Intestinal crypts were entirely male or entirely female. Villi contained columns of male or female epithelial cells, consistent with upward migration of cells from the crypts which surround them. Within the exocrine pancreas, acini were entirely male or entirely female, consistent with patterning from a single stem/progenitor cell. Pancreatic islets contained a mixture of male and female cells, consistent with patterning from multiple progenitors. Male-female chimeric mice demonstrate that the adult mouse exocrine pancreatic acinus is patterned from a single stem/progenitor cell, while the endocrine pancreas arises from multiple progenitors.
doi:10.1016/j.bbrc.2008.12.104
PMCID: PMC2657659  PMID: 19116141
Mouse pancreas development; embryonic stem cell chimera; pancreatic acinus; pancreatic islet
4.  Disruption of cAMP and Prostaglandin E2 Transport by Multidrug Resistance Protein 4 Deficiency Alters cAMP-Mediated Signaling and Nociceptive Response 
Molecular pharmacology  2007;73(1):243-251.
Multidrug resistance protein 4 (MRP4; ABCC4) is a member of the MRP/ATP-binding cassette family serving as a transmembrane transporter involved in energy-dependent efflux of anticancer/antiviral nucleotide agents and of physiological substrates, including cyclic nucleotides and prostaglandins (PGs). Phenotypic consequences of mrp4 deficiency were investigated using mrp4-knockout mice and derived immortalized mouse embryonic fibroblast (MEF) cells. Mrp4 deficiency caused decreased extracellular and increased intracellular levels of cAMP in MEF cells under normal and forskolin-stimulated conditions. Mrp4 deficiency and RNA interference-mediated mrp4 knockdown led to a pronounced reduction in extracellular PGE2 but with no accumulation of intracellular PGE2 in MEF cells. This result was consistent with attenuated cAMP-dependent protein kinase activity and reduced cyclooxygenase-2 (Cox-2) expression in mrp4-deficient MEF cells, suggesting that PG synthesis is restrained along with a lack of PG transport caused by mrp4 deficiency. Mice lacking mrp4 exhibited no outward phenotypes but had a decrease in plasma PGE metabolites and an increase in inflammatory pain threshold compared with wild-type mice. Collectively, these findings imply that mrp4 mediates the efflux of PGE2 and concomitantly modulates cAMP mediated signaling for balanced PG synthesis in MEF cells. Abrogation of mrp4 affects the regulation of peripheral PG levels and consequently alters inflammatory nociceptive responses in vivo.
doi:10.1124/mol.107.039594
PMCID: PMC2780335  PMID: 17959714
5.  Positive influence of AP-2α transcription factor on cadherin gene expression and differentiation of the ocular surface 
The family of transcription factors Activating protein-2 (AP-2) are known to play important roles in numerous developmental events, including those associated with differentiation of stratified epithelia. However, to date, the influence of the AP-2 genes on endogenous gene expression in the stratified epithelia and how this affects differentiation has not been well defined. The following study examines the detailed expression of the AP-2α and AP-2β proteins in the stratified epithelia of the ocular surface, including that in the cornea and developing eyelids. The effect of altered levels of the AP-2α gene on ocular surface differentiation was also examined using a corneal epithelial cell line and AP-2α chimeric mice. Immunolocalization studies revealed that, while AP-2β was broadly expressed throughout all cell layers of the stratified corneal epithelium, AP-2α expression was confined to cell compartments more basally located. AP-2α was also highly expressed in the less differentiated cell layers of the eyelid epidermis. Overexpression of the AP-2α gene in the corneal cell line, SIRC, resulted in a dramatic change in cell phenotype including a clumping growth behavior that was distinct from the smooth monolayer of the parent cell line. Accompanying this change was an up-regulation in levels of the cell adhesion molecule, N-cadherin. Examination of the ocular surface of AP-2α chimeric mice, derived from a mixed population of AP-2α−/− and AP-2α+/+, revealed that a down-regulation in E-cadherin expression is correlated with location of the AP-2α−/− null cells. Together, these findings demonstrate that AP-2α participates in regulating differentiation of the ocular surface through induction in cadherin expression.
PMCID: PMC2517417  PMID: 12694203
ocular surface; cell adhesion; transcription factors; AP-2; cornea; eyelids; differentiation

Results 1-5 (5)