Inhibition of gap junctional Intercellular communication in WB-F344 rat liver epithelial cells by triphenyltin chloride through MAPK and PI3-kinase pathways

<p>Abstract</p> <p>Background</p> <p>Organotin compounds (OTCs) have been widely used as stabilizers in the production of plastic, agricultural pesticides, antifoulant plaints and wood preservation. The toxicity of triphenyltin (TPT) compounds was known for their embryotoxic, neurotoxic, genotoxic and immunotoxic effects in mammals. The carcinogenicity of TPT was not well understood and few studies had discussed the effects of OTCs on gap junctional intercellular communication (GJIC) of cells.</p> <p>Method</p> <p>In the present study, the effects of triphenyltin chloride (TPTC) on GJIC in WB-F344 rat liver epithelial cells were evaluated, using the scrape-loading dye transfer technique.</p> <p>Results</p> <p>TPTC inhibited GJIC after a 30-min exposure in a concentration- and time-dependent manner. Pre-incubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 and PI3K inhibitor LY294002 decreased substantially the inhibition of GJIC by TPTC. After WB-F344 cells were exposed to TPTC, phosphorylation of Cx43 increased as seen in Western blot analysis.</p> <p>Conclusions</p> <p>These results show that TPTC inhibits GJIC in WB-F344 rat liver epithelial cells by altering the Cx43 protein expression through both MAPK and PI3-kinase pathways.</p

High-dose Immunoglobulin Infusion for Thrombotic Thrombocytopenic Purpura Refractory to Plasma Exchange and Steroid Therapy

The outcomes of the treatment of thrombotic thrombocytopenic purpura (TTP) have been shown to be improved by the administration of plasma exchange. However, treatment options are currently limited for cases refractory to plasma exchange. The autoantibodies that block the activity of ADAMTS13 have been demonstrated to play a role in the pathogenesis of TTP; therefore, high-dose immunoglobulin, which can neutralize these autoantibodies, may be useful for refractory TTP. However, successful treatment with high-dose immunoglobulin for TTP refractory to plasma exchange and corticosteroids has yet to be reported in Korea. Herein, we describe a refractory case which was treated successfully with high-dose immunoglobulin. A 29-year-old male diagnosed with TTP failed to improve after plasma exchange coupled with additional high-dose corticosteroid therapy. As a salvage treatment, we initiated a 7-day regimen of high-dose immunoglobulin (400 mg/kg) infusions, which resulted in a complete remission, lasting up to the last follow-up at 18 months. High-dose immunoglobulin may prove to be a useful treatment for patients refractory to plasma exchange; it may also facilitate recovery and reduce the need for plasma exchange

BIOSMILE web search: a web application for annotating biomedical entities and relations

BIOSMILE web search (BWS), a web-based NCBI-PubMed search application, which can analyze articles for selected biomedical verbs and give users relational information, such as subject, object, location, manner, time, etc. After receiving keyword query input, BWS retrieves matching PubMed abstracts and lists them along with snippets by order of relevancy to protein–protein interaction. Users can then select articles for further analysis, and BWS will find and mark up biomedical relations in the text. The analysis results can be viewed in the abstract text or in table form. To date, BWS has been field tested by over 30 biologists and questionnaires have shown that subjects are highly satisfied with its capabilities and usability. BWS is accessible free of charge at http://bioservices.cse.yzu.edu.tw/BWS

Neurochemical Properties of the Synapses in the Pathways of Orofacial Nociceptive Reflexes

The brainstem premotor neurons of the facial nucleus (VII) and hypoglossal (XII) nucleus can integrate orofacial nociceptive input from the caudal spinal trigeminal nucleus (Vc) and coordinate orofacial nociceptive reflex (ONR) responses. However, the synaptoarchitectures of the ONR pathways are still unknown. In the current study, we examined the distribution of GABAergic premotor neurons in the brainstem local ONR pathways, their connections with the Vc projections joining the brainstem ONR pathways and the neurochemical properties of these connections. Retrograde tracer fluoro-gold (FG) was injected into the VII or XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the Vc. Immunofluorescence histochemical labeling for inhibitory/excitatory neurotransmitters combined with BDA/FG tracing showed that GABAergic premotor neurons were mainly distributed bilaterally in the ponto-medullary reticular formation with an ipsilateral dominance. Some GABAergic premotor neurons made close appositions to the BDA-labeled fibers coming from the Vc, and these appostions were mainly distributed in the parvicellular reticular formation (PCRt), dorsal medullary reticular formation (MdD), and supratrigeminal nucleus (Vsup). We further examined the synaptic relationships between the Vc projecting fibers and premotor neurons in the VII or XII under the confocal laser-scanning microscope and electron microscope, and found that the BDA-labeled axonal terminals that made asymmetric synapses on premotor neurons showed vesicular glutamate transporter 2 (VGluT2) like immunoreactivity. These results indicate that the GABAergic premotor neurons receive excitatory neurotransmission from the Vc and may contribute to modulating the generation of the tonic ONR

Advances in research on the use of biochar in soil for remediation: a review

Purpose: Soil contamination mainly from human activities remains a major environmental problem in the contemporary world. Significant work has been undertaken to position biochar as a readily-available material useful for the management of contaminants in various environmental media notably soil. Here, we review the increasing research on the use of biochar in soil for the remediation of some organic and inorganic contaminants.  Materials and methods: Bibliometric analysis was carried out within the past 10 years to determine the increasing trend in research related to biochar in soil for contaminant remediation. Five exemplar contaminants were reviewed in both laboratory and field-based studies. These included two inorganic (i.e., As and Pb) and three organic classes (i.e., sulfamethoxazole, atrazine, and PAHs). The contaminants were selected based on bibliometric data and as representatives of their various contaminant classes. For example, As and Pb are potentially toxic elements (anionic and cationic, respectively), while sulfamethoxazole, atrazine, and PAHs represent antibiotics, herbicides, and hydrocarbons, respectively.  Results and discussion: The interaction between biochar and contaminants in soil is largely driven by biochar precursor material and pyrolysis temperature as well as some characteristics of the contaminants such as octanol-water partition coefficient (KOW) and polarity. The structural and chemical characteristics of biochar in turn determine the major sorption mechanisms and define biochar’s suitability for contaminant sorption. Based on the reviewed literature, a soil treatment plan is suggested to guide the application of biochar in various soil types (paddy soils, brownfield, and mine soils) at different pH levels (4–5.5) and contaminant concentrations ( 50 mg kg−1).  Conclusions: Research on biochar has grown over the years with significant focus on its properties, and how these affect biochar’s ability to immobilize organic and inorganic contaminants in soil. Few of these studies have been field-based. More studies with greater focus on field-based soil remediation are therefore required to fully understand the behavior of biochar under natural circumstances. Other recommendations are made aimed at stimulating future research in areas where significant knowledge gaps exist

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Kinesin 3 and cytoplasmic dynein mediate interkinetic nuclear migration in neural stem cells.

Radial glial progenitor cells exhibit bidirectional cell cycle-dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migration and neurogenesis and suggest that a cell cycle-dependent switch between distinct microtubule motors drives interkinetic nuclear migration

Asymmetric centrosome inheritance maintains neural progenitors in the neocortex. Nature 461: 947–955

Abstract Asymmetric divisions of radial glial progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. While differentiating cells leave the VZ to constitute the future neocortex, renewing radial glial progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny. Centrosome duplication in dividing radial glial progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glial progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of Ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results suggest that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glial progenitors in the developing mammalian neocortex. Radial glial cells constitute a major population of neural progenitor cells that occupy the proliferative ventricular zone (VZ) in the developing mammalian neocortex 1-3. In addition to their well-characterized function as a scaffold in supporting neuronal migration 4, radial Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials should be addressed to S.-H. Shi ([email protected]). Author Contributions X.W. and S.-H.S. conceived the project. X.W. performed most of the experiments. J.-W.T., W.-N.L. and R.B.V. contributed to the time-lapse imaging experiment and J.H.I. contributed to the characterization of Kaede-Centrin1 co-localization and in utero photoconversion procedure. X. W. and S.-H. S. analyzed data, interpreted results and wrote the manuscript. All authors edited the manuscript. HHS Public Access Author Manuscript Author Manuscript Author Manuscript Author Manuscript glial cells display interkinetic nuclear oscillation and proliferate extensively at the luminal surface of the VZ (i.e. the VZ surface). During the peak phase of neurogenesis (around embryonic day 13 to 18, E13-E18, in mice), they predominantly undergo asymmetric division to self-renew while simultaneously giving rise either directly to a neuron, or to an intermediate progenitor cell (IPC) that subsequently divides symmetrically to produce neurons 5-8. While differentiating progeny progressively migrate away from the VZ to form the cortical plate (CP) -the future neocortex, renewing radial glial progenitors remain in the VZ for subsequent divisions. The distinct migratory behaviour of radial glial progenitors and their differentiating progeny is fundamental to the proper development of the mammalian neocortex; however, little is known about the basis of these behavioural differences. Centrosomes, the main microtubule-organizing centres (MTOCs) in animal cells 9, play an important role in many cell processes, particularly during cell division 10 and cell migration 11-13. All normal animal cells initially inherit one centrosome, consisting of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). The two centrioles differ in their structure and function 9, 14, 15. The older &quot;mother&quot; centriole, which is formed at least one and a half generations earlier, possesses appendages/satellites that bear specific proteins, such as Cenexin/Odf2 16, 17 and Ninein 18-20, and anchor microtubules and support ciliogenesis 9, 21. In contrast, the younger &quot;daughter&quot; centriole, which is formed during the preceding S phase, lacks these structures. Full acquisition of appendages/satellites by the daughter centriole is not achieved until at least one and a half cell cycles later 22, 23. During each cell cycle, the centrosome replicates once in a semi-conservative manner 24, resulting in the formation of two centrosomes -one of which retains the original old mother centriole (i.e. the mother centrosome) while the other receives the new mother centriole (i.e. the daughter centrosome) 14, 15. This intrinsic asymmetry in the centrosome has recently been demonstrated to be important for proper spindle orientation during the division of male germline stem cells 25, 26 and neuroblasts 27, 28 in Drosophila, although female germline stem cells appear to divide normally in the absence of centrioles/centrosomes 29. These studies indicate a critical role for the differential behaviour of centrosomes with differently aged mother centrioles in asymmetric division of the progenitor/stem cells 30-33, although it remains unclear whether proper behaviour and development of the progenitor/stem cells and their differentiating daughter cells depend on centrosome asymmetry. Asymmetric division of radial glial progenitors accounts for nearly all neurogenesis in the developing mammalian neocortex 5-8. Three out of four autosomal recessive primary microcephaly (MCPH) genes identified so far encode centrosomal components 34, suggesting that proper neocortical neurogenesis and development entail a tight regulation of the centrosome 35, which is so far poorly understood. To address these issues, we investigated centrosome regulation during the peak phase of mammalian neocortical neurogenesis ( Centriole and centrosome asymmetry To examine centrosome behaviour, we introduced a plasmid encoding Centrin1, a central component of the centriole, fused with enhanced green fluorescent protein (EGFP-Centrin1) into the developing neocortex of E13.5 mouse embryos by in utero electroporation ( Author Manuscript Author Manuscript Author Manuscript Author Manuscript with γ-Tubulin, a centrosomal marker To identify the cell types harbouring EGFP-Centrin1-labelled centrosomes, we coelectroporated a plasmid encoding DsRedexpress (DsRedex), a red fluorescence protein that diffuses throughout cells and thereby reveals their morphology The distinct positioning of the centrosome in radial glial progenitors versus their differentiating progeny prompted us to ask whether the centrosomes in these two cell populations/types are different. To explore this, we electroporated a plasmid encoding Ninein, a mature centriole-specific protein that localizes to appendages/satellites 18, 19, fused with EGFP (EGFP-Ninein) together with a plasmid encoding Centrin1 fused to DsRedex (DsRedex-Centrin1) into the developing mouse neocortex at E13.5. As expected, both EGFP-Ninein and DsRedex-Centrin1 formed dot-like structures and co-localized to the centrosomes, especially those at the VZ surface, as identified by an antibody to the integral centrosomal protein Pericentrin1 Having found that the centrosomes in dividing radial glial progenitors exhibit asymmetry in their maturity, we next asked whether this centrosome asymmetry is related to the distinct In vivo pulse-chase labelling of centrosomes To test this, we first developed an assay to explicitly distinguish between the centrosome containing the old mother centriole and the centrosome containing the new mother centriole in the developing neocortex in vivo ( To carry out this assay in the developing neocortex in vivo, we developed an in utero photoconversion procedure and combined it with in utero electroporation We found that one day after photo-conversion (E13.5-E14.5(PC)-E15.5), around 95% of centrosomes contained both red and green fluorescent centrioles (indicated by yellow colour in the merged image) ( Asymmetric segregation and inheritance of centrosomes Having successfully distinguished the centrosomes with differently aged mother centrioles in the developing neocortex in vivo, we next examined their distribution to determine whether they are asymmetrically segregated. Remarkably, we found that more than 76% of centrosomes with the new mother centriole (i.e. only green fluorescent) were located in the IZ and the CP, whereas around 78% of centrosomes with the old mother centriole (i.e. both green and red fluorescent) were located in the VZ in addition to the SVZ The asymmetric segregation of centrosomes suggests differential regulation of the duplicated centrosomes in dividing radial glial progenitors. To gather further evidence for this, we carried out time-lapse imaging experiments to monitor the behaviour of centrosomes with differently aged mother centrioles in dividing radial glial progenitors at the VZ surface in situ The distinct behaviour of the centrosomes suggests that they are differentially inherited by the two daughter cells embarking on different routes of fate specification and development. Based on their behaviour, we postulated that the centrosome with the new mother centriole is largely inherited by differentiating cells, such as neurons, while the centrosome with the old mother centriole that remains located at the VZ is mostly inherited by radial glial progenitors. Indeed, we found that two days after photo-conversion (E13.5-E14.5(PC)-E16.5) the centrosomes with the new mother centriole, marked by green fluorescence alone, were mostly associated with cells expressing TUJ1, a differentiating neuronal marker, in the CP and the IZ Asymmetric centrosome inheritance maintains progenitors Our data thus far show that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glial progenitors in the developing neocortex. We next tested whether the selective inheritance of the centrosome with the old mature mother centriole by radial glial progenitors is necessary for their maintenance at the VZ. Should this be the case, given that Ninein is an essential component of the appendage/satellite structures specific to the mature centriole, we predicted that removal of Ninein, which prevents centriole maturation 19, 42, would disrupt asymmetric segregation of centrosomes with differently aged mother centrioles and impair the maintenance of radial glial progenitors in the developing neocortex. To test this, we developed short hairpin RNA (shRNA) sequences against Ninein that effectively suppressed its expression (Ninein shRNAs, Author Manuscript Author Manuscript Author Manuscript Author Manuscript asymmetric segregation of centrosomes with differently aged mother centrioles labelled with Kaede-Centrin1 in the developing neocortex ( To further characterize the extent to which removal of Ninein leads to a depletion of radial glial progenitors, we next examined the fate specification of cells expressing either control or Ninein shRNA ( Previous studies showed that the carboxyl-terminus of Ninein is responsible for its localization to the centriole and expression of this region displaces endogenous protein at the centriole 43. Interestingly, we found that, similar to removal of Ninein, expression of the carboxyl-terminus of Ninein (Ninein-Cter) led to a premature depletion of radial glial progenitor cells from the VZ In summary, the results presented here suggest that the centrosomes with differently aged centrioles in asymmetrically dividing radial glial progenitors exhibit different behaviour and are differentially inherited by the two daughter cells during the peak phase of mammalian neocortical neurogenesis Page 7 Nature. Author manuscript; available in PMC 2010 April 15. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Drosophila male germline stem cells (GSCs) and neuroblasts 25-28. Our findings suggest that this type of asymmetric centrosome regulation may be a general feature of asymmetric cell division across species 30-33. Furthermore, our findings provide new insight into centrosome regulation in the developing mammalian neocortex, which has been linked to the pathogenesis of human microcephaly 34, 44. Centrosomes with differently aged mother centrioles differ in their protein composition and thereby in their biophysical properties, such as microtubule anchorage activity 9, 15 and the capability to mediate ciliogenesis 21, 23, 45. In this study, we found that Ninein, an appendage/satellite-specific protein required for centriole maturation, localized differently to the duplicated centrosomes in radial glial progenitors in late mitosis. Interestingly, another appendage/satellite-specific protein Cenexin/Odf2 was recently found to be asymmetrically localized to centrosomes in sister cells after mitosis; moreover, the cell receiving the more mature old mother centriole usually grew a primary cilium first 21. The asymmetric inheritance of centrosomes with distinct biophysical properties may thereby differentially regulate the behaviour and development of the daughter cells that receive them. For example, given that primary cilia play essential roles in a number of signal transduction pathways, including Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signalling, the asynchrony in cilium formation could differentially influence the ability of the two daughter cells to respond to environmental signals and thereby their behaviour and fate specification. Also, the strong microtubule anchorage activity associated with the centrosome retaining the older mother centriole would facilitate its anchorage to a specific site (e.g. the VZ surface), thereby tethering the cell that inherits it. Indeed, we found that disruption of centriole maturation by removing Ninein not only impairs asymmetric segregation of centrosomes, but also depletes radial glial progenitors from the VZ, a proliferative niche in the developing mammalian neocortex. Aside from their participation in microtubule organization and ciliogenesis, centrosomes associate with messenger RNAs (mRNAs) 46 and membrane-bound organelles such as the Golgi and recycling endosomes and regulate protein degradation 47, 48, thereby raising the possibility that asymmetric centrosome inheritance might contribute to proper segregation of cell fate determinants to the two daughter cells of asymmetrically dividing progenitor/stem cells. Methods summary In utero electroporation and photo-conversion In utero electroporation of the plasmids (e.g. EGFP-Centrin1) was performed as previously described 49, 50. For in utero photo-conversion, a similar surgical procedure was carried out as for in utero electroporation. The forebrain of the embryos that received electroporation was exposed to a brief (about three to five minutes) exposure of light at 350-400 nm while in the uterus. All procedures for animal handling and usage were approved by our institutional research animal resource centre (RARC). Brain section, immunohistochemistry and imaging Brains were fixed at the desired developmental stages and coronal sections were prepared using a vibratome (Leica Microsystems). Immunohistochemistry were performed as Author Manuscript Author Manuscript Author Manuscript Author Manuscript previously described 50. Images were acquired using a confocal laser scanning microscope (FV1000, Olympus) and analyzed using FluoView (Olympus), Volocity (Improvision) and Photoshop (Adobe Systems). Data were presented as mean and s.e.m. and statistical differences were determined using the nonparametric tests (Mann-Whitney-Wilcoxon test for two groups of data and Kruskal-Wallis test for three or more groups of data). Cortical slice culture and time-lapse imaging Cortical slice cultures were prepared and time-lapse imaging was acquired as previously described 12. Images were analyzed using MetaMorph (Molecular Devices) and Photoshop (Adobe Systems). Methods Plasmids and in utero electroporation and photo-conversion shRNA-a (5′-GCAGAAGGCCAGCTGAGGT-3′), -b (GGCCGAGATCCGGCACTTG), -c (GCTTCAATTCAGACAATGG). All sense and anti-sense oligos were purchased from Sigma. Annealed oligos were cloned into the HpaI and XhoI sites of the Lentiviral vector pLL3.7, which contains a separate CMV promoter that drives expression of EGFP 50. In this study, mouse Ninein shRNA-c was primarily used after extensive characterization to demonstrate that it specifically suppressed Ninein protein expression and function. For siRNA experiments, synthetic oligos against Ninein and control were purchased from Santa Cruz Biotechnology (sc-61196). Human Ninein cDNA, which differs from mouse Ninein cDNA in the shRNA-c targeting region and is thereby insensitive to Ninein shRNA-c expression, was used for the rescue experiment. The amino-terminus (nucleotides 1-1120) and carboxyl-terminus (nucleotides 5623-6339) of mouse Ninein were amplified by PCR and cloned into the EcoRI and NotI sites of pCAG-IRES-EGFP. All plasmids were confirmed by sequencing. In utero electroporation was performed as previously described 49, 50. In brief, a timed pregnant CD-1 mouse at 13.5 days of gestation (E13.5) or a rat at E16.5 was anesthetized, the uterine horns were exposed, and ~1 μl of plasmid DNA (1-3 μg/μl) mixed with Fast Green (Sigma,) was manually microinjected through the uterus into the lateral ventricle, using a bevelled and calibrated glass micropipette (Drummond Scientific). For electroporation, five 50 ms pulses of 40-50 mV with a 950 ms interval were delivered across the uterus with two 9-mm electrode paddles positioned on either side of the head (BTX, ECM830). For in utero photo-conversion, a similar surgical procedure was carried out as for in utero electroporation. The forebrain of the embryos that received Wang et al. Page 9 Nature. Author manuscript; available in PMC 2010 April 15. Author Manuscript Author Manuscript Author Manuscript Author Manuscript electroporation was exposed to a brief (about three to five minutes) exposure of light at 350-400 nm while in the uterus. Throughout these surgical procedures, the uterus was constantly bathed with warm phosphate buffered saline (PBS, pH 7.4). After the procedure, the uterus was placed back in the abdominal cavity and the wound was surgically sutured. The animal was then placed in a 28°C recovery incubator under close monitoring until it recovered and resumed normal activity. All procedures for animal handling and usage were approved by our institutional research animal resource centre (RARC). Brain sectioning and confocal imaging and analysis Embryos were removed and transcardially perfused with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde (PFA) in PBS (pH 7.4). For cell cycle exit analysis, electroporated embryos were exposed to bromodeoxyuridine (BrdU, ~50-100 mg/kg body weight) for 24 hours prior to sacrifice. Brains were dissected out and coronal sections were prepared using a vibratome (Leica Microsystems). For immunohistochemistry, sections were incubated for one hour at room temperature in a blocking solution (10% normal goat or donkey serum as appropriate, 0.1% Triton X-100, and 0.2% gelatin in PBS), followed by incubation with the primary antibodies overnight at 4°C. Sections were then washed in 0.1% Triton X-100 in PBS and incubated with the appropriate secondary antibody for one to two hours at room temperature. The primary antibodies used were: rabbit polyclonal anti-γ-Tubulin (Sigma, 1:500), mouse monoclonal anti-Pericentrin1 (BD Biosciences, 1:1000), mouse monoclonal anti-β-III Tubulin (clone TUJ1) (Covance, 1:500), rabbit polyclonal anti-Pax6 (Covance, 1:500), rabbit polyclonal anti-Tbr2 (Millipore/Chemicon, 1:500), rat monoclonal anti-BrdU (Abcam, 1:400), mouse polyclonal anti-Ki67 (Novus Biological, 1:200), rabbit polyclonal anti-GLAST (Invitrogen, 1:400), rabbit polyclonal anti-phospho-Histone 3 (Millipore/ Upstate, 1:1000), mouse monoclonal anti-phospho-H2AX (Millipore/Upstate, 1:250) and rabbit polyclonal anti-cleaved Caspase 3 (Cell Signalling Technology, 1:250). Secondary antibodies used were: goat or donkey anti-mouse or anti-rabbit Alexa-546 and Alexa-647 conjugated antibodies (Invitrogen/Molecular Probes, 1:500). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen/Molecular Probes). Images were acquired with an Olympus FV1000 confocal microscope, and analyzed with FluoView (Olympus), Volocity (Improvision), and Photoshop (Adobe Systems). Data are presented as mean ± standard error of the mean (s.e.m.) and nonparametric tests (Mann-Whitney-Wilcoxon test for two groups of data and Kruskal-Wallis test for three or more groups of data) were used for statistical significance estimation. Cortical slice culture and time-lapse imaging Cortical slice cultures were prepared and time-lapse imaging was acquired as previously described 12. About 12 hours after in utero electroporation, embryos were removed and the brain was extracted into ice-cold artificial cerebro-spinal fluid (ACSF) containing (in mM): 125 NaCl, 5 KCl, 1.25 NaH 2 PO 4 , 1 MgSO 4 , 2 CaCl 2 , 25 NaHCO 3 and 20 glucose; pH 7.4, 310 mOsm/L. Brains were embedded in 4% low-melting agarose in ACSF and sectioned at 400 μm using a vibratome (Leica microsystems). Brain slices that contained Kaede- Author Manuscript Author Manuscript Author Manuscript Author Manuscript Centrin1-and mPlum-expressing cells were transferred onto a slice culture insert (Millicell) in a glass-bottom petri dish (MatTek Corporation) with culture medium containing (by volume): 66% BME, 25% Hanks, 5%