Jin Pu

Schizophrenia-related neural and behavioral phenotypes in transgenic mice expressing truncated Disc1.

Disrupted-in-Schizophrenia-1 (DISC1), identified by positional cloning of a balanced translocation (1;11) with the breakpoint in intron 8 of a large Scottish pedigree, is associated with a range of neuropsychiatric disorders including schizophrenia. To model this mutation in mice, we have generated Disc1tr transgenic mice expressing 2 copies of truncated Disc1 encoding the first 8 exons using a bacterial artificial chromosome (BAC). With this partial simulation of the human situation, we have discovered a range of phenotypes including a series of novel features not previously reported. Disc1tr transgenic mice display enlarged lateral ventricles, reduced cerebral cortex, partial agenesis of the corpus callosum, and thinning of layers II/III with reduced neural proliferation at midneurogenesis. Parvalbumin GABAergic neurons are reduced in the hippocampus and medial prefrontal cortex, and displaced in the dorsolateral frontal cortex. In culture, transgenic neurons grow fewer and shorter neurites. Behaviorally, transgenic mice exhibit increased immobility and reduced vocalization in depression-related tests, and impairment in conditioning of latent inhibition. These abnormalities in Disc1tr transgenic mice are consistent with findings in severe schizophrenia.

Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo

It has long been known that cells can be induced to migrate by the application of small d.c. electric fields (EFs), a phenomenon referred to as galvanotaxis. We recently reported some significant effects of electric signals of physiological strength in guiding cell migration and wound healing. We present here protocols to apply an EF to cells or tissues cultured in an electrotactic chamber. The chamber can be built to allow controlled medium flow to prevent the potential development of chemical gradients generated by the EFs. It can accommodate cells on planar culture or tissues in 3D gels. Mounted on an inverted microscope, this setup allows close and well-controlled observation of cellular responses to electric signals. As similar EFs are widely present during development and wound healing, this experimental system can be used to simulate and study cellular and molecular responses to electric signals in these events.

Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field

Directed cell migration is essential for tissue formation, inflammation, and wound healing. Chemotaxis plays a major role in these situations and is underpinned by asymmetric intracellular signaling. Endogenous electric fields (EFs) are common where cell movement occurs, such as in wound healing, and cells respond to electric field gradients by reorienting and migrating directionally (galvanotaxis/electrotaxis). We show that a physiological EF redistributed both EGF (epidermal growth factor) receptors and detergent‐insoluble membrane lipids asymmetrically, leading to cathodal polarization and enhanced activation of the MAP kinase, ERK1/2. This induced leading‐edge actin polymerization in directionally migrating mammalian epithelial cells. Inhibiting the EGF receptor‐MAP kinase signaling pathway significantly decreased leading edge actin asymmetry and directional migration. We propose a model in which EF‐polarized membrane lipid domains and EGF receptors cause asymmetric signaling through MAP kinase, which drives directional cell migration. A comparison is made with the mechanisms underpinning chemotaxis.

EGF receptor signalling is essential for electric-field-directed migration of breast cancer cells

The mechanisms by which cancer cells migrate to metastasise are not fully understood. Breast cancers are accompanied by electrical depolarisation of tumour epithelial cells. The electrical changes can be detected on the skin and are used to differentiate malignant from benign breast tumours. Could the electrical signals play a role in metastasis by promoting tumour cell migration? We report that electric fields stimulate and direct migration of human breast cancer cells. Importantly, these effects were more significant in highly metastatic tumour cells than in low metastatic tumour cells. Electric-field-enhanced directional migration correlates well with the expression level of EGF receptor (EGFR/ErbB1). To confirm this, we transfected low metastatic clone MTC cells with human ErbB1, which significantly increased the electrotactic response. Inhibition of ErbB1 completely abolished the directional response of MTLn3 cells to an electric field. Transfection of MTLn3 cells and MDA-MB-435 cells with expression vectors for ErbB family members ErbB1, ErbB2 and ErbB3 also significantly enhanced EF-induced migration. These results suggest that electric signals might play a role in metastasis of breast cancers by enhancing cell migration through the ErbB-signalling pathway.

E-cadherin plays an essential role in collective directional migration of large epithelial sheets

In wound healing and development, large epithelial sheets migrate collectively, in defined directions, and maintain tight cell–cell adhesion. This type of movement ensures an essential function of epithelia, a barrier, which is lost when cells lose connection and move in isolation. Unless wounded, epithelial sheets in cultures normally do not have overall directional migration. Cell migration is mostly studied when cells are in isolation and in the absence of mature cell–cell adhesion; the mechanisms of the migration of epithelial sheets are less well understood. We used small electric fields (EFs) as a directional cue to instigate and guide migration of epithelial sheets. Significantly, cells in monolayer migrated far more efficiently and directionally than cells in isolation or smaller cell clusters. We demonstrated for the first time the group size-dependent directional migratory response in several types of epithelial cells. Gap junctions made a minimal contribution to the directional collective migration. Breaking down calcium-dependent cell–cell adhesion significantly reduced directional sheet migration. Furthermore, E-cadherin blocking antibodies abolished migration of cell sheets. Traction force analysis revealed an important role of forces that cells in the leading rows exert on the substratum. With EF, the traction forces of the leading edge cells coordinated in directional re-orientation. Our study thus identifies a novel mechanism—E-cadherin dependence and coordinated traction forces of leading cells in collective directional migration of large epithelial sheets.

Direct visualization of a stratified epithelium reveals that wounds heal by unified sliding of cell sheets.

Observing cells in their original niche is a key link between the information gleaned from planar culture and in vivo physiology and pathology. A new approach combining the transparency of the cornea, Hoffman modulation optics, and digital imaging allowed movements of individual corneal cells to be viewed directly in situ. 3‐Dimensional time‐lapse movies imaging unstained cells within the stratified corneal epithelium during wound healing were made. Tracking cell movements dynamically provided a definitive answer to the long‐standing question: does a stratified epithelium heal by “sliding” of cell sheets as a coherent unit or do individual cells “leap frog” each other at the wound margin? A wound in the corneal epithelium healed primarily by sliding of the whole epithelium, with ~95% of cells moving with similar speed and trajectories and with little change in their relative position. Only 5% of cells changed layers, with equal proportions moving up or down. Epithelial healing in situ occurred in three phases: a latency, migration, and reconstruction phase. This model provides a unique system to study the behaviors of individual cells in their original niche. It shows that cells slide into a wound as a unified unit to heal a stratified epithelium.—Zhao, M., Song, B., Pu, J., Forrester, J. V., McCaig, C. D. Direct visualization of a stratified epithelium reveals that wounds heal by unified sliding of cell sheets. FASEB J. 17, 397–406 (2003)

Golgi polarization in a strong electric field

Directional cell migration requires proper cell polarization. The redistribution of the Golgi apparatus is an important event in the polarization and migration of many types of cells, as a polarized Golgi supplies membrane components for leading edge protrusion. Direct current electric fields induce directional cell migration in a wide variety of cells. Here we show that electric fields of 300 mV/mm induce robust Golgi polarization and directional cell migration in CHO cells. Asymmetric Src and PI 3-kinase signalling as well as actin polymerization are essential for electric field-induced Golgi polarization and directional cell migration. The Golgi polarizes at the same time as cells change morphology and migrate directionally in response to an electric field. Golgi polarization in turn significantly reinforces and maintains optimal electrotaxis. It is not known whether electrical signals, when contradicting other directional cues, are still able to polarize cells and direct cell migration. Most strikingly, Golgi polarization and cell migration simply follow the direction of an applied electric field and ignore all other cues generated by wounding a monolayer of CHO cells. Thus, an electric field of 300 mV/mm is the predominant cue to polarize the Golgi and direct cell migration mediated by PI 3-kinase and Src signalling.

Endogenous electric currents might guide rostral migration of neuroblasts

Mechanisms that guide directional migration of neuroblasts from the subventricular zone (SVZ) are not well understood. We report here that endogenous electric currents serve as a guidance cue for neuroblast migration. We identify the existence of naturally occurring electric currents (1.5±0.6 μA/cm2, average field strength of ∼3 mV/mm) along the rostral migration path in adult mouse brain. Electric fields of similar strength direct migration of neuroblasts from the SVZ in culture and in brain slices. The purinergic receptor P2Y1 mediates this migration. The results indicate that naturally occurring electric currents serve as a new guidance mechanism for rostral neuronal migration.

Derivation of homogeneous GABAergic neurons from mouse embryonic stem cells

Embryonic stem cells (ESCs) promise an unlimited source of defined cells for cell transplantation therapy, while protocols for derivation of homogeneous populations of desirable cell types are yet to be developed and/or refined. Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, and disturbed GABAergic signaling is associated with a host of neurological conditions. We developed a simple ES cell differentiation protocol which led to the production of uniform GABAergic neurons in approximately 2 weeks. The differentiation protocol involved treatment of embryoid bodies (EBs) with high concentrations (10(-5)-10(-)(4) M) of all-trans-retinoic acid (RA) for 3 days. After plating these EBs on attached dishes in neural supportive medium, 93-96% of the cells became GABA-positive neurons in 7-11 days. These cells also expressed immature neuronal markers with voltage-gated delayed rectifier potassium currents, suggesting that they were immature GABAergic neurons. The technology may have implications for modeling and treatment of GABAergic signaling-related diseases and injuries.

ß-adrenergic receptor agonists delay while antagonists accelerate epithelial wound healing: Evidence of an endogenous adrenergic network within the corneal epithelium

Wound healing is a complex and well‐orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of ß2‐adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. ß‐adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A‐mediated mechanism and decrease both electric field‐directed migration and corneal wound healing. Conversely, ß‐adrenergic receptor antagonists accelerate CEC migration, enhance electric field‐mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (ß‐adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro‐motogenic effect of the ß‐adrenergic antagonist is blockade of the ß2‐adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs. J. Cell. Physiol. 211: 261–272, 2007.