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1.  Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture 
Cell  2013;153(7):1510-1525.
The molecular mechanisms underlying the axon arborization of mammalian neurons are poorly understood but are critical for the establishment of functional neural circuits. We identified a pathway defined by two kinases, LKB1 and NUAK1, required for cortical axon branching in vivo. Conditional deletion of LKB1 after axon specification or knockdown of NUAK1 drastically reduced axon branching in vivo whereas their overexpression was sufficient to increase axon branching. The LKB1-NUAK1 pathway controls mitochondria immobilization in axons. Using manipulation of Syntaphilin, a protein necessary and sufficient to arrest mitochondrial transport specifically in the axon, we demonstrate that the LKB1-NUAK1 kinase pathway regulates axon branching by promoting mitochondria immobilization. Finally, we show that LKB1 and NUAK1 are necessary and sufficient to immobilize mitochondria specifically at nascent presynaptic sites. Our results unravel a link between presynaptic mitochondrial capture and axon branching.
PMCID: PMC3729210  PMID: 23791179
2.  Nuclear localization of Prickle2 is required to establish cell polarity during early mouse embryogenesis 
Developmental Biology  2012;364(2):138-148.
The establishment of trophectoderm (TE) manifests as the formation of epithelium, and is dependent on many structural and regulatory components that are commonly found and function in many epithelial tissues. However, the mechanism of TE formation is currently not well understood. Prickle1 (Pk1), a core component of the planar cell polarity (PCP) pathway, is essential for epiblast polarization before gastrulation, yet the roles of Pk family members in early mouse embryogenesis are obscure. Here we found that Pk2−/− embryos died at E3.0–3.5 without forming the blastocyst cavity and not maintained epithelial integrity of TE. These phenotypes were due to loss of the apical-basal (AB) polarity that underlies the asymmetric redistribution of microtubule networks and proper accumulation of AB polarity components on each membrane during compaction. In addition, we found GTP-bound active form of nuclear RhoA was decreased in Pk2−/− embryos during compaction. We further show that the first cell fate decision was disrupted in Pk2−/− embryos. Interestingly, Pk2 localized to the nucleus from the 2-cell to around the 16-cell stage despite its cytoplasmic function previously reported. Inhibiting farnesylation blocked Pk2’s nuclear localization and disrupted AB cell polarity, suggesting that Pk2 farnesylation is essential for its nuclear localization and function. The cell polarity phenotype was efficiently rescued by nuclear but not cytoplasmic Pk2, demonstrating the nuclear localization of Pk2 is critical for its function.
PMCID: PMC3299875  PMID: 22333836
3.  Gbx2 Directly Restricts Otx2 Expression to Forebrain and Midbrain, Competing with Class III POU Factors 
Molecular and Cellular Biology  2012;32(13):2618-2627.
Otx2 plays essential roles in rostral brain development, and its counteraction with Gbx2 has been suggested to determine the midbrain-hindbrain boundary (MHB) in vertebrates. We previously identified the FM enhancer that is conserved among vertebrates and drives Otx2 transcription in forebrain/midbrain from the early somite stage. In this study, we found that the POU homeodomain of class III POU factors (Brn1, Brn2, Brn4, and Oct6) associates with noncanonical target sequence TAATTA in the FM enhancer. MicroRNA-mediated knockdown of Brn2 and Oct6 diminished the FM enhancer activity in anterior neural progenitor cells (NPCs) differentiated from P19 cells. The class III POU factors associate with the FM enhancer in forebrain and midbrain but not in hindbrain. We also demonstrated that the Gbx2 homeodomain recognizes the same target TAATTA in the FM enhancer, and Gbx2 associates with the FM enhancer in hindbrain. Gbx2 misexpression in the anterior NPCs repressed the FM enhancer activity and inhibited Brn2 association with the enhancer, whereas Gbx2 knockdown caused ectopic Brn2 association in the posterior NPCs. These results suggest that class III POU factors and Gbx2 share the same target site, TAATTA, in the FM enhancer and that their region-specific binding restricts Otx2 expression at the MHB.
PMCID: PMC3434480  PMID: 22566684
4.  A Mouse Reporter Line to Conditionally Mark Nuclei and Cell Membranes for In Vivo Live-Imaging 
Genesis (New York, N.Y. : 2000)  2011;49(7):570-578.
Live-imaging is an essential tool to visualize live cells and monitor their behaviors during development. This technology demands a variety of mouse reporter lines, each uniquely expressing a fluorescent protein. Here, we developed an R26R-RG reporter mouse line that conditionally and simultaneously expresses mCherry and EGFP in nuclei and plasma membranes, respectively, from the Rosa26 locus. The intensity and resolution of mCherry nuclear localization and EGFP membrane localization were demonstrated to be sufficient for live-imaging with embryos that express RG (mCherry and EGFP) ubiquitously and specifically in fetal Sertoli cells. The conditional R26R-RG reporter mouse line should be a useful tool for labeling nuclei and membranes simultaneously in distinct cell populations.
PMCID: PMC3466090  PMID: 21504045
Rosa26 locus; conditional cell labeling; dual cell labeling; nuclear labeling; membrane labeling
5.  EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial–mesenchymal transition 
The Journal of Cell Biology  2008;182(6):1217-1230.
EPB41L5 belongs to the band 4.1 superfamily. We investigate here the involvement of EPB41L5 in epithelial–mesenchymal transition (EMT) during mouse gastrulation. EPB41L5 expression is induced during TGFβ-stimulated EMT, whereas silencing of EPB41L5 by siRNA inhibits this transition. In EPB41L5 mutants, cell–cell adhesion is enhanced, and EMT is greatly impaired during gastrulation. Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency. Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression. However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm. We find that EPB41L5 binds p120ctn through its N-terminal FERM domain, inhibiting p120ctn–E-cadherin binding. EPB41L5 overexpression causes E-cadherin relocalization into Rab5-positive vesicles in epithelial cells. At the same time, EPB41L5 binds to paxillin through its C terminus, enhancing integrin/paxillin association, thereby stimulating focal adhesion formation.
PMCID: PMC2542480  PMID: 18794329
6.  Sfrp Controls Apicobasal Polarity and Oriented Cell Division in Developing Gut Epithelium 
PLoS Genetics  2009;5(3):e1000427.
Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut.
Author Summary
The gastrointestinal tract is generated from the primitive gut tube during embryogenesis. The primitive gut differentiates regionally along the cephalocaudal axis. Individual regions simultaneously acquire specific morphologies through morphogenetic mechanisms. The regional specification of the gut tube is controlled by cross-talk between the mesenchyme and epithelium. However, the morphogenetic mechanisms governing gut formation remain poorly understood. Secreted Frizzled-related protein (Sfrp) is an inhibitor of the Wnt pathway, members of which are expressed in the developing gut. A deficiency of Sfrp genes (Sfrp1, Sfrp2, and Sfrp5) results in reduction of fore-stomach length in mice. During normal fore-stomach formation, cell division is oriented along the cephalocaudal axis; in contrast, reduced fore-stomach length in Sfrps-deficient mice is associated with the divergence of oriented cell division in tubular epithelial cells. Thus, oriented cell division is one of the essential components in fore-stomach morphogenesis. In addition, Sfrps-deficient small intestine epithelium fails to maintain proper apicobasal polarity. We also found that Wnt5a-inactivation leads to a phenotype similar to that induced by Sfrps-deficiency in the developing gut, and that Sfrp1 inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling is required for oriented cell division and that it modulates apicobasal polarity in gut epithelium during organ elongation.
PMCID: PMC2649445  PMID: 19300477
7.  Functional Roles of Otx2 Transcription Factor in Postnatal Mouse Retinal Development▿ † 
Molecular and Cellular Biology  2007;27(23):8318-8329.
We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/−; Crx−/− retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx−/− retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/−; Crx−/− retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.
PMCID: PMC2169187  PMID: 17908793
8.  Glucokinase and IRS-2 are required for compensatory β cell hyperplasia in response to high-fat diet–induced insulin resistance 
Journal of Clinical Investigation  2007;117(1):246-257.
Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of β cell–specific Gck (Gck+/–) causes impaired insulin secretion to glucose, although the animals have a normal β cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked β cell hyperplasia, whereas Gck+/– mice demonstrated decreased β cell replication and insufficient β cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet–fed Gck+/– mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet–fed wild-type mice compared with those fed standard chow and reduced expression in HF diet–fed Gck+/– mice compared with those of HF diet–fed wild-type mice. HF diet–fed Irs2+/– mice failed to show a sufficient increase in β cell mass, and overexpression of Irs2 in β cells of HF diet–fed Gck+/– mice partially prevented diabetes by increasing β cell mass. These results suggest that Gck and Irs2 are critical requirements for β cell hyperplasia to occur in response to HF diet–induced insulin resistance.
PMCID: PMC1716196  PMID: 17200721
9.  Immunocytochemical Analyses and Targeted Gene Disruption of GTPBP1 
Molecular and Cellular Biology  2000;20(17):6195-6200.
We previously identified a gene encoding a putative GTPase, GTPBP1, which is structurally related to elongation factor 1α, a key component of protein biosynthesis machinery. The primary structure of GTPBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1. Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that GTPBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages. Immunofluorescence analyses revealed that GTPBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages. To investigate the physiological role of GTPBP1, we used targeted gene disruption in embryonic stem cells to generate GTPBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in GTPBP1-deficient mice may be due to functional compensation by GTPBP2, a molecule we recently identified which is similar to GTPBP1 in structure and tissue distribution.
PMCID: PMC86094  PMID: 10938096
10.  Extracellular Matrix Survival Signals Transduced by Focal Adhesion Kinase Suppress p53-mediated Apoptosis  
The Journal of Cell Biology  1998;143(2):547-560.
In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C λ/ι and cytosolic phospholipase A2. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.
PMCID: PMC2132850  PMID: 9786962
fibronectin; survival; FAK; p53; apoptosis
11.  Circadian regulation of intracellular G-protein signalling mediates intercellular synchrony and rhythmicity in the suprachiasmatic nucleus 
Nature Communications  2011;2:327-.
Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell–cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gαi, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.
Circadian rhythm is controlled by the suprachiasmatic nucleus and the mechanisms that control the rhythm are largely undiscovered. In this study, a G protein regulator, RGS16, is shown to be involved in the production of cyclic AMP that is required for the suprachiasmatic nucleus to maintain rhythm
PMCID: PMC3112533  PMID: 21610730

Results 1-11 (11)