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An effective combination of multi-omic datasets can enhance our understanding of complex biological phenomena. To build a context-dependent network with multiple omic layers, i.e., a trans-omic network, we perform phosphoproteomics, transcriptomics, proteomics, and metabolomics of murine liver for 4 h after insulin administration and integrate the resulting time series. Structural characteristics and dynamic nature of the network are analyzed to elucidate the impact of insulin. Early and prominent changes in protein phosphorylation and persistent and asynchronous changes in mRNA and protein levels through non-transcriptional mechanisms indicate enhanced crosstalk between phosphorylation-mediated signaling and protein expression regulation. Metabolic response shows different temporal regulation with transient increases at early time points across categories and enhanced response in the amino acid and nucleotide categories at later time points as a result of process convergence. This extensive and dynamic view of the trans-omic network elucidates prominent regulatory mechanisms that drive insulin responses through intricate interlayer coordination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Although the postpartum period is suggested to provide an ideal opportunity for interventions to prevent hazardous drinking, evidence on the associations of education and income with hazardous drinking during this period is limited, including in Japan., Methods: We analyzed data from 11,031 women who participated in the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study in Japan. Hazardous drinking was defined as ethanol intake of ≥20 g/day 1 year after delivery. We conducted multiple logistic regression analyses to examine whether educational attainment or equivalent household income was associated with hazardous drinking, adjusting for age, parity, drinking status during pregnancy, work status, postpartum depression, breastfeeding, and income/education. We also conducted stratified analyses by income and education groups., Results: The prevalence of hazardous drinking 1 year after delivery was 3.6%. Lower education was associated with hazardous drinking; the odds ratio (95% confidence interval) of high school education or lower compared with university education or higher was 2.17 (1.59-2.98). Lower income was also associated with hazardous drinking, but this association disappeared after further adjustments for education; the odds ratios (95% confidence intervals) of the lowest compared with highest level of income were 1.42 (1.04-1.94) and 1.12 (0.81-1.54), respectively. A significant interaction was detected; lower education and lower income were associated with increased risks of hazardous drinking only in a lower income group and lower education group, respectively., Conclusions: Postpartum women with lower education and lower income had higher risks of hazardous drinking in Japan.

Although many coronavirus 2019 patients have experienced persistent symptoms and a long-term decline in quality of life after discharge, the details of these persistent symptoms and the effect of early rehabilitation are still unclear. We conducted a single-center, retrospective observational study to investigate the prevalence of persistent symptoms three months after discharge from the intensive care unit by checking the medical records. All patients received an early mobilization program. Four out of 13 patients (31%) had postintensive care syndrome. No patients had muscle weakness, and 11 patients (85%) returned to their previous work. However, psychiatric disorder, such as anxiety (23%) and posttraumatic stress disorder (15%), were observed. Eleven patients claimed persistent symptoms, including fatigue and numbness in the extremities. Our results suggest that the implementation of an early rehabilitation program plays some role in preventing muscle weakness and that decreasing psychiatric disorders should be a next target of patient care in the intensive care unit.

Introduction: Evaluation of the safety of taking lamotrigine (LTG) during lactation in breastfed infants varies according to the information sources. As it is possible that prescribers may avoid prescribing LTG despite of it being one of the essential drugs, more information needs to be accumulated to facilitate its use. Materials and Methods: We retrospectively compared the safety of LTG during the lactation period in 20 pairs of mothers and infants with 20 pairs as the control group. Results: The mean dose of LTG in 20 mothers was 161.1 mg/day (range: 50-400 mg/day). None of the infants showed a neonatal withdrawal syndrome score of 2 or more up to 1 month after delivery. Although drowsiness ( n  = 3), skin rash ( n  = 11), jaundice ( n  = 8), heart murmur ( n  = 1), poor suckling ( n  = 1), and retractive breathing ( n  = 1) were observed in infants, none of these adverse events were serious and the infants recovered. Nineteen of 20 pairs could continue lactation until 1 month after delivery. One pair discontinued breastfeeding because of pain in the mother's nipples. All pairs could continue maternal medication. We then compared the results with those of the control group. There were no significant differences in the presence of adverse events between the LTG and control groups. Conclusion: These data suggest that taking low to moderate doses of LTG during the lactation period might be relatively safe, at least for a period of 1 month after delivery.

Blood and lymphatic vessels surrounding the heart develop through orchestrated processes from cells of different origins. In particular, cells around the outflow tract which constitute a primordial transient vasculature, referred to as aortic subepicardial vessels, are crucial for the establishment of coronary artery stems and cardiac lymphatic vessels. Here, we revealed that the epicardium and pericardium-derived Semaphorin 3E (Sema3E) and its receptor, PlexinD1, play a role in the development of the coronary stem, as well as cardiac lymphatic vessels. In vitro analyses demonstrated that Sema3E may demarcate areas to repel PlexinD1-expressing lymphatic endothelial cells, resulting in proper coronary and lymphatic vessel formation. Furthermore, inactivation of Sema3E-PlexinD1 signaling improved the recovery of cardiac function by increasing reactive lymphangiogenesis in an adult mouse model of myocardial infarction. These findings may lead to therapeutic strategies that target Sema3E-PlexinD1 signaling in coronary artery diseases., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.

Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics.

Brain structures are diverse among species despite the essential molecular machinery of neurogenesis being common. Recent studies have indicated that differences in the mechanical properties of tissue may result in the dynamic deformation of brain structure, such as folding. However, little is known about the correlation between mechanical properties and species-specific brain structures. To address this point, a comparative analysis of mechanical properties using several animals is required. For a systematic measurement of the brain stiffness of remotely maintained animals, we developed a novel strategy of tissue-stiffness measurement using glyoxal as a fixative combined with atomic force microscopy. A comparison of embryonic and juvenile mouse and songbird brain tissue revealed that glyoxal fixation can maintain brain structure as well as paraformaldehyde (PFA) fixation. Notably, brain tissue fixed by glyoxal remained much softer than PFA-fixed brains, and it can maintain the relative stiffness profiles of various brain regions. Based on this method, we found that the homologous brain regions between mice and songbirds exhibited different stiffness patterns. We also measured brain stiffness in other amniotes (chick, turtle, and ferret) following glyoxal fixation. We found stage-dependent and species-specific stiffness in pallia among amniotes. The embryonic chick and matured turtle pallia showed gradually increasing stiffness along the apico-basal tissue axis, the lowest region at the most apical region, while the ferret pallium exhibited a catenary pattern, that is, higher in the ventricular zone, the inner subventricular zone, and the cortical plate and the lowest in the outer subventricular zone. These results indicate that species-specific microenvironments with distinct mechanical properties emerging during development might contribute to the formation of brain structures with unique morphology., (Copyright © 2020 Iwashita, Nomura, Suetsugu, Matsuzaki, Kojima and Kosodo.)

Endosome maturation depends on membrane contact sites (MCSs) formed between endoplasmic reticulum (ER) and endolysosomes (LyLEs). The mechanism underlying lipid supply for this process and its pathophysiological relevance remains unclear, however. Here, we identify PDZD8-the mammalian ortholog of a yeast ERMES subunit-as a protein that interacts with protrudin, which is located at ER-LyLE MCSs. Protrudin and PDZD8 promote the formation of ER-LyLE MCSs, and PDZD8 shows the ability to extract various lipids from the ER. Overexpression of both protrudin and PDZD8 in HeLa cells, as well as their depletion in mouse primary neurons, impairs endosomal homeostasis by inducing the formation of abnormal large vacuoles reminiscent of those apparent in spastin- or REEP1-deficient neurons. The protrudin-PDZD8 system is also essential for the establishment of neuronal polarity. Our results suggest that protrudin and PDZD8 cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, thereby maintaining neuronal polarity and integrity.

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic β-amyloid (Aβ) peptide generated from proteolytic cleavage of β-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin ( PSEN ) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aβ generation in vitro , and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism in vivo remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP in vivo We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic β-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aβ in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aβ-dependent pathogenicity in AD., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Gotoh et al.)

Background: During development, Cajal-Retzius (CR) cells are the first generated and essential pioneering neurons that control neuronal migration and arealization in the mammalian cortex. CR cells are derived from specific regions within the telencephalon, that is, the pallial septum in the rostromedial cortex, the pallial-subpallial boundary, and the cortical hem (CH) in the caudomedial cortex. However, the molecular mechanism underlying the generation of CR cell subtypes in distinct regions of origin is poorly understood., Results: We found that double-sex and mab-3 related transcription factor (Dmrt) genes, that is, Dmrta1 and Dmrt3, were expressed in the progenitor domains that produce CR cells. The number of CH-derived CR cells was severely decreased in Dmrt3 mutants, especially in Dmrta1 and Dmrt3 double mutants. The reduced production of the CR cells was consistent with the developmental impairment of the CH structures in the medial telencephalon from which the CR cells are produced., Conclusion: Dmrta1 and Dmrt3 cooperatively regulate patterning of the CH structure and production of the CR cells from the CH during cortical development., (© 2020 Wiley Periodicals, Inc.)

The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.

Neural stem cells, called radial glia, maintain epithelial structure during the early neocortical development. The prevailing view claims that when radial glia first proliferate, their symmetric divisions require strict spindle orientation; its perturbation causes precocious neurogenesis and apoptosis. Here, we show that despite this conventional view, radial glia at the proliferative stage undergo normal symmetric divisions by regenerating an apical endfoot even if it is lost by oblique divisions. We found that the Notch-R-Ras-integrin β1 pathway promotes the regeneration of endfeet, whose leading edge bears ectopic adherens junctions and the Par-polarity complex. However, this regeneration ability gradually declines during the subsequent neurogenic stage and hence oblique divisions induce basal translocation of radial glia to form the outer subventricular zone, a hallmark of the development of the convoluted brain. Our study reveals that endfoot regeneration is a temporally changing cryptic property, which controls the radial glial state and its shift is essential for mammalian brain size expansion.

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1 + lineage-specific deletion of Prox1 resulted in disrupted LYVE1 + vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3 + and/or Prox1 + cells that overlapped with the Isl1 + pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here, we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, Dmrt3 and Dmrta2 show a medial high /lateral low expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity., Competing Interests: KK, SY, IF, YO, AS, TS, FM No competing interests declared, (© 2019, Kono et al.)

The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C 9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C 9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry. IMPORTANCE Phanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C 9-15 ) and fatty acids (C 9-15 ) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications., (Copyright © 2018 American Society for Microbiology.)

The cerebral cortex is a highly organized structure whose development depends on diverse progenitor cell types, namely apical radial glia, intermediate progenitors, and basal radial glia cells, which are responsible for the production of the correct neuronal output. In recent years, these progenitor cell types have been deeply studied, particularly basal radial glia and their role in cortical expansion and gyrification. We review here a broad series of factors that regulate progenitor behavior and daughter cell fate. We first describe the different neuronal progenitor types, emphasizing the differences between lissencephalic and gyrencephalic species. We then review key factors shown to influence progenitor proliferation versus differentiation, discussing their roles in progenitor dynamics, neuronal production, and potentially brain size and complexity. Although spindle orientation has been considered a critical factor for mode of division and daughter cell output, we discuss other features that are emerging as crucial for these processes such as organelle and cell cycle dynamics. Additionally, we highlight the importance of adhesion molecules and the polarity complex for correct cortical development. Finally, we briefly discuss studies assessing progenitor multipotency and its possible contribution to the production of specific neuronal populations. This review hence summarizes recent aspects of cortical progenitor cell biology, and pinpoints emerging features critical for their behavior., (© 2018 International Society for Neurochemistry.)

In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gα i subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gα i in vitro , comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation., (© 2017 The Authors.)

Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.

Neural stem cells go through a sequence of timely regulated gene expression and pattern of division mode to generate diverse neurons during brain development. During vertebrate cerebral cortex development, neural stem cells begin with proliferative symmetric divisions, subsequently undergo neurogenic asymmetric divisions, and finally gliogenic divisions. In this review, we explore the relationship between stem cell versus neural fate specification and the division mode. Specifically, we discuss recent findings on the mechanisms of asymmetric divisions, division mode, and developmental progression of neural progenitor identity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation., (© 2017. Published by The Company of Biologists Ltd.)

Organoids mimicking the formation of the brain cortex have been demonstrated to be powerful tools for developmental studies as well as pathological investigations of brain malformations. Here, we report an integrated approach for the quantification of temporal neural production (neurogenic rate) in organoids derived from embryonic brains. Spherical tissue fragments with polarized cytoarchitectures were incubated in multiple cavities arranged in a polymethylmethacrylate chip. The time-dependent neurogenic rate in the organoids was monitored by the level of EGFP under the promoter of Tbr2, a transcription factor that is transiently expressed in neural fate-committed progenitors during corticogenesis. Importantly, our monitoring system exhibited a quick response to DAPT, a drug that promotes neural differentiation. Furthermore, we successfully quantified the temporal neurogenic rate in a large number of organoids by applying image processing that semi-automatically recognized the positions of organoids and measured their signal intensities from sequential images. Taken together, we provide a strategy to quantitate the neurogenic rate in brain organoids in a time-dependent manner, which will also be a potent method for monitoring organoid formation and drug activity in other tissue types., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation., Competing Interests: The authors have declared that no competing interests exist.

The van der Waals compound Xe(N 2 ) 2 with a C15 Laves structure was successfully synthesised at pressures greater than 4.4 GPa. We found that, at 10 GPa, the structure reversibly transforms from a cubic to a tetragonal phase. Further compression results in changes of Xe-N compound, which could result in the enhancement of orbital interactions between the xenon and nitrogen atoms.

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Mutations in a synaptic organizing pathway contribute to autism. Autism-associated mutations in MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) are thought to reduce excitatory/inhibitory transmission. However, we show that mutation of Mdga2 elevates excitatory transmission, and that MDGA2 blocks neuroligin-1 interaction with neurexins and suppresses excitatory synapse development. Mdga2(+/-) mice, modeling autism mutations, demonstrated increased asymmetric synapse density, mEPSC frequency and amplitude, and altered LTP, with no change in measures of inhibitory synapses. Behavioral assays revealed an autism-like phenotype including stereotypy, aberrant social interactions, and impaired memory. In vivo voltage-sensitive dye imaging, facilitating comparison with fMRI studies in autism, revealed widespread increases in cortical spontaneous activity and intracortical functional connectivity. These results suggest that mutations in MDGA2 contribute to altered cortical processing through the dual disadvantages of elevated excitation and hyperconnectivity, and indicate that perturbations of the NRXN-NLGN pathway in either direction from the norm increase risk for autism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as 'ectodomain shedding', is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo.

The embryonic brain is one of the tissues most vulnerable to ionizing radiation. In this study, we showed that ionizing radiation induces apoptosis in the neural progenitors of the mouse cerebral cortex, and that the surviving progenitor cells subsequently develop a considerable amount of supernumerary centrosomes. When mouse embryos at Day 13.5 were exposed to γ-rays, brains sizes were reduced markedly in a dose-dependent manner, and these size reductions persisted until birth. Immunostaining with caspase-3 antibodies showed that apoptosis occurred in 35% and 40% of neural progenitor cells at 4 h after exposure to 1 and 2 Gy, respectively, and this was accompanied by a disruption of the apical layer in which mitotic spindles were positioned in unirradiated mice. At 24 h after 1 Gy irradiation, the apoptotic cells were completely eliminated and proliferation was restored to a level similar to that of unirradiated cells, but numerous spindles were localized outside the apical layer. Similarly, abnormal cytokinesis, which included multipolar division and centrosome clustering, was observed in 19% and 24% of the surviving neural progenitor cells at 48 h after irradiation with 1 and 2 Gy, respectively. Because these cytokinesis aberrations derived from excess centrosomes result in growth delay and mitotic catastrophe-mediated cell elimination, our findings suggest that, in addition to apoptosis at an early stage of radiation exposure, radiation-induced centrosome overduplication could contribute to the depletion of neural progenitors and thereby lead to microcephaly.

During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.

Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties., Significance Statement: Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking.Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to direct the migration, differentiation, circuit integration, and maintenance programs within distinct subtypes of CGE-derived interneurons., (Copyright © 2015 the authors 0270-6474/15/3512870-21$15.00/0.)

Studies of human brain malformations, such as lissencephaly and double cortex, have revealed the importance of neuronal migration during cortical development. Afadin, a membrane scaffolding protein, regulates the formation of adherens junctions (AJs) and cell migration to form and maintain tissue structures. Here, we report that mice with dorsal telencephalon-specific ablation of afadin gene exhibited defects similar to human double cortex, in which the heterotopic cortex was located underneath the normotopic cortex. The normotopic cortex of the mutant mice was arranged in the pattern similar to the cortex of the control mice, while the heterotopic cortex was disorganized. As seen in human patients, double cortex in the mutant mice was formed by impaired neuronal migration during cortical development. Genetic ablation of afadin in the embryonic cerebral cortex disrupted AJs of radial glial cells, likely resulting in the retraction of the apical endfeet from the ventricular surface and the dispersion of radial glial cells from the ventricular zone to the subventricular and intermediate zones. These results indicate that afadin is required for the maintenance of AJs of radial glial cells and that the disruption of AJs might cause an abnormal radial scaffold for neuronal migration. In contrast, the proliferation or differentiation of radial glial cells was not significantly affected. Taken together, these findings indicate that afadin is required for the maintenance of the radial glial scaffold for neuronal migration and that the genetic ablation of afadin leads to the formation of double cortex., (Copyright © 2015 Elsevier B.V. All rights reserved.)

During mammalian brain development, neural progenitor cells undergo symmetric proliferative divisions followed by asymmetric neurogenic divisions. The division mode of these self-renewing progenitors, together with the cell fate of their progeny, plays critical roles in determining the number of neurons and, ultimately, the size of the adult brain. In the past decade, remarkable progress has been made toward identifying various types of neuronal progenitors. Recent technological advances in live imaging and genetic manipulation have enabled us to link dynamic cell biological events to the molecular mechanisms that control the asymmetric divisions of self-renewing progenitors and have provided a fresh perspective on the modes of division of these progenitors. In addition, comparison of progenitor repertoires between species has provided insight into the expansion and the development of the complexity of the brain during mammalian evolution., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre–lox-based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2+ and Vav1+ compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

This study aims to investigate the effectiveness of monolithic translucent TZP with different colors and porcelain-layered TZP by evaluating their colors and strengths. Different mixing ratios of Zpex to Zpex-Yellow as translucent TZP, conventional opaque TZP (TZ-3YB-E) (Tosoh, Tokyo) as a control, and veneering porcelain (CERABIEN ZR, body porcelain, Noritake, Tokyo) with shade A3 as a typical shade. Disk-shaped specimens of 13 mm diameter and 1.5 mm thickness were prepared. These specimens were observed under reflected and transmitted light, and the translucency parameter (TP) values were measured. Strength was also evaluated with flexural strength in a biaxial bending test. The TP values of the monolithic TZP, Zpex100>Zpex70>Zpex50>TZ3YB, were larger in this order. The flexural strength of all the monolithic TZP showed approximately 1,000 MPa. It is suggested that colored translucent TZP is clinically useful when used as monolithic restorations.

The coordinated polarization of neighboring cells within the plane of the tissue, known as planar cell polarity (PCP), is a recurring theme in biology. It is required for numerous developmental processes for the form and function of many tissues and organs across species. The genetic pathway regulating PCP was first discovered in Drosophila, and an analogous but distinct pathway is emerging in vertebrates. It consists of membrane protein complexes known as core PCP proteins that are conserved across species. Here we report that the over-expression of the murine Ankrd6 (mAnkrd6) gene that shares homology with Drosophila core PCP gene diego causes a typical PCP phenotype in Drosophila, and mAnkrd6 can rescue the loss of function of diego in Drosophila. In mice, mAnkrd6 protein is asymmetrically localized in cells of the inner ear sensory organs, characteristic of components of conserved core PCP complexes. The loss of mAnkrd6 causes PCP defects in the inner ear sensory organs. Moreover, canonical Wnt signaling is significantly increased in mouse embryonic fibroblasts from mAnkrd6 knockout mice in comparison to wild type controls. Together, these results indicated that mAnkrd6 is a functional homolog of the Drosophila diego gene for mammalian PCP regulation and act to suppress canonical Wnt signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.

Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.

Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.

Aurora-A kinase (AurA) is a key regulator of cellular processes involving microtubules. It has also been implicated in actin-dependent events, but the mechanisms that underlie the processes are not fully understood. Here we provide genetic and biochemical evidence suggesting that AurA negatively regulates Drok, the only known Rho-kinase orthologue in Drosophila. AurA directly phosphorylates Drok in vitro, and the overexpression of the nonphosphorylatable forms of Drok in vivo causes similar, but much stronger effects than that of wild-type Drok. The defects induced by the nonphosphorylatable forms of Drok are compensated by reducing the function of myosin downstream. Thus, phosphorylation of Drok by AurA normally suppresses Drok activity. We propose that AurA directly regulates actin-dependent processes by phosphorylating Rho-kinase., (Copyright © 2013 The authors. Published by Elsevier Inc. All rights reserved.)

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.