X
Forgot Password

If you have forgotten your password you can enter your email here and get a temporary password sent to your email.

Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568

RRID:AB_2534013

Restricted Presence of POU6F2 in Human Corneal Endothelial Cells Uncovered by Extension of the Promoter-level Expression Atlas.

  • Yoshihara M
  • EBioMedicine
  • 2018 Jul 9

Literature context:


Abstract:

Corneal endothelial cells (CECs) are essential for maintaining the clarity of the cornea. Because CECs have limited proliferative ability, interest is growing in their potentially therapeutic regeneration from pluripotent stem cells. However, the molecular mechanisms of human CEC differentiation remain largely unknown. To determine the key regulators of CEC characteristics, here we generated a comprehensive promoter-level expression profile of human CECs, using cap analysis of gene expression (CAGE) with a single molecule sequencer. Integration with the FANTOM5 promoter-level expression atlas, which includes transcriptome profiles of various human tissues and cells, enabled us to identify 45 promoters at 28 gene loci that are specifically expressed in CECs. We further discovered that the expression of transcription factor POU class 6 homeobox 2 (POU6F2) is restricted to CECs, and upregulated during human CEC differentiation, suggesting that POU6F2 is pivotal to terminal differentiation of CECs. These CEC-specific promoters would be useful for the assessment of fully differentiated CECs derived from pluripotent stem cells. These findings promote the development of corneal regenerative medicine.

Funding information:
  • NIMH NIH HHS - R01 MH094714(United States)

Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

  • Nakano Y
  • Cell
  • 2018 Jun 25

Literature context:


Abstract:

The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.

Funding information:
  • NIMH NIH HHS - 5 F32 MH064339-03(United States)

Arginine Methylation by PRMT2 Controls the Functions of the Actin Nucleator Cobl.

  • Hou W
  • Dev. Cell
  • 2018 Apr 23

Literature context:


Abstract:

The complex architecture of neuronal networks in the brain requires tight control of the actin cytoskeleton. The actin nucleator Cobl is critical for neuronal morphogenesis. Here we reveal that Cobl is controlled by arginine methylation. Coprecipitations, coimmunoprecipitations, cellular reconstitutions, and in vitro reconstitutions demonstrated that Cobl associates with the protein arginine methyltransferase PRMT2 in a Src Homology 3 (SH3) domain-dependent manner and that this promotes methylation of Cobl's actin nucleating C-terminal domain. Consistently, PRMT2 phenocopied Cobl functions in both gain- and loss-of-function studies. Both PRMT2- and Cobl-promoted dendritogenesis relied on methylation. PRMT2 effects require both its catalytic domain and SH3 domain. Cobl-mediated dendritic arborization required PRMT2, complex formation with PRMT2, and PRMT2's catalytic activity. Mechanistic studies reveal that Cobl methylation is key for Cobl actin binding. Therefore, arginine methylation is a regulatory mechanism reaching beyond controlling nuclear processes. It also controls a major, cytosolic, cytoskeletal component shaping neuronal cells.

Funding information:
  • NHLBI NIH HHS - HL24415(United States)

Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression.

  • Seino T
  • Cell Stem Cell
  • 2018 Mar 1

Literature context:


Abstract:

Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression.

Spatial-Temporal Lineage Restrictions of Embryonic p63+ Progenitors Establish Distinct Stem Cell Pools in Adult Airways.

  • Yang Y
  • Dev. Cell
  • 2018 Mar 26

Literature context:


Abstract:

Basal cells (BCs) are p63-expressing multipotent progenitors of skin, tracheoesophageal and urinary tracts. p63 is abundant in developing airways; however, it remains largely unclear how embryonic p63+ cells contribute to the developing and postnatal respiratory tract epithelium, and ultimately how they relate to adult BCs. Using lineage-tracing and functional approaches in vivo, we show that p63+ cells arising from the lung primordium are initially multipotent progenitors of airway and alveolar lineages but later become restricted proximally to generate the tracheal adult stem cell pool. In intrapulmonary airways, these cells are maintained immature to adulthood in bronchi, establishing a rare p63+Krt5- progenitor cell population that responds to H1N1 virus-induced severe injury. Intriguingly, this pool includes a CC10 lineage-labeled p63+Krt5- cell subpopulation required for a full H1N1-response. These data elucidate key aspects in the establishment of regionally distinct adult stem cell pools in the respiratory system, potentially with relevance to other organs.

Funding information:
  • Intramural NIH HHS - ZIA HL006151-02(United States)
  • NCI NIH HHS - R01 CA112403()
  • NCI NIH HHS - R01 CA193455()
  • NHLBI NIH HHS - R35 HL135834()
  • NIAID NIH HHS - HHSN272201400008C()

Overlapping Role of SCYL1 and SCYL3 in Maintaining Motor Neuron Viability.

  • Kuliyev E
  • J. Neurosci.
  • 2018 Feb 7

Literature context:


Abstract:

Members of the SCY1-like (SCYL) family of protein kinases are evolutionarily conserved and ubiquitously expressed proteins characterized by an N-terminal pseudokinase domain, centrally located HEAT repeats, and an overall disorganized C-terminal segment. In mammals, 3 family members encoded by genes Scyl1, Scyl2, and Scyl3 have been described. Studies have pointed to a role for SCYL1 and SCYL2 in regulating neuronal function and viability in mice and humans, but little is known about the biological function of SCYL3. Here, we show that the biochemical and cell biological properties of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. Specifically, although lack of Scyl3 in mice has no apparent effect on embryogenesis and postnatal life, it accelerates the onset of the motor neuron disorder caused by Scyl1 deficiency. Growth abnormalities, motor dysfunction, hindlimb paralysis, muscle wasting, neurogenic atrophy, motor neuron degeneration, and loss of large-caliber axons in peripheral nerves occurred at an earlier age in Scyl1/Scyl3 double-deficient mice than in Scyl1-deficient mice. Disease onset also correlated with the mislocalization of TDP-43 in spinal motor neurons, suggesting that SCYL1 and SCYL3 regulate TDP-43 proteostasis. Together, our results demonstrate an overlapping role for SCYL1 and SCYL3 in vivo and highlight the importance the SCYL family of proteins in regulating neuronal function and survival. Only male mice were used in this study.SIGNIFICANCE STATEMENTSCYL1 and SCYL2, members of the SCY1-like family of pseudokinases, have well-established roles in neuronal function. Herein, we uncover the role of SCYL3 in maintaining motor neuron viability. Although targeted disruption of Scyl3 in mice had little or no effect on embryonic development and postnatal life, it accelerated disease onset associated with the loss of Scyl1, a novel motor neuron disease gene in humans. Scyl1 and Scyl3 double-deficient mice had neuronal defects characteristic of amyotrophic lateral sclerosis, including TDP-43 pathology, at an earlier age than did Scyl1-deficient mice. Thus, we show that SCYL1 and SCYL3 play overlapping roles in maintaining motor neuronal viability in vivo and confirm that SCYL family members are critical regulators of neuronal function and survival.

Funding information:
  • NCI NIH HHS - P50 CA093459(United States)

A Pixel-Encoder Retinal Ganglion Cell with Spatially Offset Excitatory and Inhibitory Receptive Fields.

  • Johnson KP
  • Cell Rep
  • 2018 Feb 6

Literature context:


Abstract:

The spike trains of retinal ganglion cells (RGCs) are the only source of visual information to the brain. Here, we genetically identify an RGC type in mice that functions as a pixel encoder and increases firing to light increments (PixON-RGC). PixON-RGCs have medium-sized dendritic arbors and non-canonical center-surround receptive fields. From their receptive field center, PixON-RGCs receive only excitatory input, which encodes contrast and spatial information linearly. From their receptive field surround, PixON-RGCs receive only inhibitory input, which is temporally matched to the excitatory center input. As a result, the firing rate of PixON-RGCs linearly encodes local image contrast. Spatially offset (i.e., truly lateral) inhibition of PixON-RGCs arises from spiking GABAergic amacrine cells. The receptive field organization of PixON-RGCs is independent of stimulus wavelength (i.e., achromatic). PixON-RGCs project predominantly to the dorsal lateral geniculate nucleus (dLGN) of the thalamus and likely contribute to visual perception.

Funding information:
  • Biotechnology and Biological Sciences Research Council - MC_U105161047(United Kingdom)
  • NEI NIH HHS - R01 EY023341()
  • NEI NIH HHS - R01 EY026978()
  • NEI NIH HHS - R01 EY027411()

Identification of Neurotensin Receptor Expressing Cells in the Ventral Tegmental Area across the Lifespan.

  • Woodworth HL
  • eNeuro
  • 2018 Feb 22

Literature context:


Abstract:

Neurotensin (Nts) promotes activation of dopamine (DA) neurons in the ventral tegmental area (VTA) via incompletely understood mechanisms. Nts can signal via the G protein-coupled Nts receptors 1 and 2 (NtsR1 and NtsR2), but the lack of methods to detect NtsR1- and NtsR2-expressing cells has limited mechanistic understanding of Nts action. To overcome this challenge, we generated dual recombinase mice that express FlpO-dependent Cre recombinase in NtsR1 or NtsR2 cells. This strategy permitted temporal control over recombination, such that we could identify NtsR1- or NtsR2-expressing cells and determine whether their distributions differed between the developing and adult brain. Using this system, we found that NtsR1 is transiently expressed in nearly all DA neurons and in many non-DA neurons in the VTA during development. However, NtsR1 expression is more restricted within the adult brain, where only two thirds of VTA DA neurons expressed NtsR1. By contrast, NtsR2 expression remains constant throughout lifespan, but it is predominantly expressed within glia. Anterograde tract tracing revealed that NtsR1 is expressed by mesolimbic, not mesocortical DA neurons, suggesting that VTA NtsR1 neurons may represent a functionally unique subset of VTA DA neurons. Collectively, this work reveals a cellular mechanism by which Nts can directly engage NtsR1-expressing DA neurons to modify DA signaling. Going forward, the dual recombinase strategy developed here will be useful to selectively modulate NtsR1- and NtsR2-expressing cells and to parse their contributions to Nts-mediated behaviors.

Funding information:
  • NIAID NIH HHS - R01 AI087528(United States)
  • NIDDK NIH HHS - F30 DK107163()
  • NIDDK NIH HHS - R01 DK103808()
  • NIGMS NIH HHS - T32 GM092715()

Opposing expression gradients of calcitonin-related polypeptide alpha (Calca/Cgrpα) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea.

  • Wu JS
  • J. Comp. Neurol.
  • 2018 Feb 15

Literature context:


Abstract:

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain-sensing C-fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C-fibers: calcitonin-related polypeptide alpha (Calca/Cgrpα), specific to pain-sensing C-fibers, and tyrosine hydroxylase (Th), specific to low-threshold mechanoreceptive C-fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., ). Whole-mount cochlear preparations from 3-week- to 2-month-old CGRPα-EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II-like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co-expression could be observed, most abundantly in the middle cochlear turn.

Funding information:
  • NIDCD NIH HHS - R01 DC006476()
  • NIDCD NIH HHS - R01 DC011741()
  • NIDCD NIH HHS - R01 DC012957()

Generation of FHL2 homozygous knockout lines from human embryonic stem cells by CRISPR/Cas9-mediated ablation.

  • Chang CW
  • Stem Cell Res
  • 2018 Jan 2

Literature context:


Abstract:

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Mutations in the FHL2 (Four and a half LIM domains protein 2) gene are associated with cardiomyopathy in patients. Here, we generated two homozygous knockout lines using CRISPR/Cas9-mediated ablation in a human embryonic stem cell (hESC) WA09 line. These knockout lines exhibit a normal karyotype without expressing FHL2 protein, while maintaining pluripotency and differentiation properties. These isogenic mutation lines will be provided as a disease model for cardiomyopathy studies and drug screening.

Funding information:
  • NCRR NIH HHS - S10RR027926(United States)

Generation of a GDE heterozygous mutation human embryonic stem cell line WAe001-A-14 by CRISPR/Cas9 editing.

  • Xu G
  • Stem Cell Res
  • 2018 Jan 9

Literature context:


Abstract:

Glycogen debranching enzyme (GDE) plays a critical role in glycogenolysis. Mutations in the GDE gene are associated with a metabolic disease known as glycogen storage disease type III (GSDIII). We generated a mutant GDE human embryonic stem cell line, WAe001-A-14, using the CRISPR/Cas9 editing system. This cell line contains a 24-nucleotide deletion within exon-13 of GDE, resulting in 8 amino acids (TRLGISSL) missing of the GDE protein from amino acid position 567 to 575. The WAe001-A-14 cell line maintains typical stem cell morphology, pluripotency and in vitro differentiation potential, and a normal karyotype.

Radial Glial Fibers Promote Neuronal Migration and Functional Recovery after Neonatal Brain Injury.

  • Jinnou H
  • Cell Stem Cell
  • 2018 Jan 4

Literature context:


Abstract:

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

Funding information:
  • NIDDK NIH HHS - R01 DK082659(United States)

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

  • Liao HK
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.

Funding information:
  • NHLBI NIH HHS - R01 HL083047(United States)
  • NHLBI NIH HHS - R01 HL123755()
  • NIAID NIH HHS - R01 AI108651()

Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination.

  • Seemann E
  • Elife
  • 2017 Dec 5

Literature context:


Abstract:

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

Funding information:
  • NHLBI NIH HHS - HL095590(United States)

Generation of a SMO homozygous knockout human embryonic stem cell line WAe001-A-16 by CRISPR/Cas9 editing.

  • Wu F
  • Stem Cell Res
  • 2017 Dec 27

Literature context:


Abstract:

The human SMO protein encoded by the smoothened (SMO) gene acts as a positive mediator for Hedgehog signaling. This pathway regulates many cellular activities, developmental morphogenesis, and tumorigenesis. Using CRISPR/Cas9 to edit human embryonic stem cell line WA01 (H1), we established a SMO mutant cell line (WAe001-A-16). This cell line has a 40bp homozygous deletion in exon 2 of SMO leading to a shift in the open reading frame and early termination at amino acid position 287. WAe001-A-16 maintains a normal karyotype, parental cell morphology, pluripotency markers, and the capacity to differentiate into all three germline layers.

Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia.

  • O'Hagan R
  • Curr. Biol.
  • 2017 Nov 20

Literature context:


Abstract:

Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia.

Funding information:
  • NIDDK NIH HHS - R01 DK059418()
  • NIDDK NIH HHS - R01 DK074746()
  • NIH HHS - R24 OD010943()

NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis.

  • Widenmaier SB
  • Cell
  • 2017 Nov 16

Literature context:


Abstract:

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.

Funding information:
  • NINDS NIH HHS - F31NS077750(United States)

Experience-Dependent Synaptic Plasticity in V1 Occurs without Microglial CX3CR1.

  • Schecter RW
  • J. Neurosci.
  • 2017 Nov 1

Literature context:


Abstract:

Brief monocular deprivation (MD) shifts ocular dominance and reduces the density of thalamic synapses in layer 4 of the mouse primary visual cortex (V1). We found that microglial lysosome content is also increased as a result of MD. Previous studies have shown that the microglial fractalkine receptor CX3CR1 is involved in synaptic development and hippocampal plasticity. We therefore tested the hypothesis that neuron-to-microglial communication via CX3CR1 is an essential component of visual cortical development and plasticity in male mice. Our data show that CX3CR1 is not required for normal development of V1 responses to visual stimulation, multiple forms of experience-dependent plasticity, or the synapse loss that accompanies MD in layer 4. By ruling out an essential role for fractalkine signaling, our study narrows the search for understanding how microglia respond to active synapse modification in the visual cortex.SIGNIFICANCE STATEMENT Microglia in the visual cortex respond to monocular deprivation with increased lysosome content, but signaling through the fractalkine receptor CX3CR1 is not an essential component in the mechanisms of visual cortical development or experience-dependent synaptic plasticity.

Funding information:
  • NEI NIH HHS - R01 EY012309()

A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication.

  • Miller CN
  • Cell Host Microbe
  • 2017 Sep 13

Literature context:


Abstract:

Many intracellular pathogens exploit host secretory trafficking to support their intracellular cycle, but knowledge of these pathogenic processes is limited. The bacterium Brucella abortus uses a type IV secretion system (VirB T4SS) to generate a replication-permissive Brucella-containing vacuole (rBCV) derived from the host ER, a process that requires host early secretory trafficking. Here we show that the VirB T4SS effector BspB contributes to rBCV biogenesis and Brucella replication by interacting with the conserved oligomeric Golgi (COG) tethering complex, a major coordinator of Golgi vesicular trafficking, thus remodeling Golgi membrane traffic and redirecting Golgi-derived vesicles to the BCV. Altogether, these findings demonstrate that Brucella modulates COG-dependent trafficking via delivery of a T4SS effector to promote rBCV biogenesis and intracellular proliferation, providing mechanistic insight into how bacterial exploitation of host secretory functions promotes pathogenesis.

Funding information:
  • NIAID NIH HHS - R01 AI129992()
  • NIAID NIH HHS - R21 AI112649()
  • NIAID NIH HHS - T32 AI007025()

Spin infection enables efficient gene delivery to muscle stem cells.

  • Kodaka Y
  • BioTechniques
  • 2017 Aug 1

Literature context:


Abstract:

Viral vector-mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts. Centrifugation force was also tested, and a spin infection protocol with centrifugation at 2800 × g for 90 min had the highest infection efficiency for primary myoblasts. We confirmed that infected muscle stem cells maintain cell proliferation and the capacity for in vitro and in vivo myogenic differentiation. Our new, efficient retroviral infection protocol for muscle stem cells can be applied to molecular biology experiments as well as translational studies.

Funding information:
  • NIAMS NIH HHS - R01 AR062142()
  • NIAMS NIH HHS - R21 AR070319()

Differentiation of Human Pluripotent Stem Cells into Colonic Organoids via Transient Activation of BMP Signaling.

  • Múnera JO
  • Cell Stem Cell
  • 2017 Jul 6

Literature context:


Abstract:

Gastric and small intestinal organoids differentiated from human pluripotent stem cells (hPSCs) have revolutionized the study of gastrointestinal development and disease. Distal gut tissues such as cecum and colon, however, have proved considerably more challenging to derive in vitro. Here we report the differentiation of human colonic organoids (HCOs) from hPSCs. We found that BMP signaling is required to establish a posterior SATB2+ domain in developing and postnatal intestinal epithelium. Brief activation of BMP signaling is sufficient to activate a posterior HOX code and direct hPSC-derived gut tube cultures into HCOs. In vitro, HCOs express colonic markers and contained colon-specific cell populations. Following transplantation into mice, HCOs undergo morphogenesis and maturation to form tissue that exhibits molecular, cellular, and morphologic properties of human colon. Together these data show BMP-dependent patterning of human hindgut into HCOs, which will be valuable for studying diseases including colitis and colon cancer.

Funding information:
  • NIAID NIH HHS - U19 AI116491()
  • NIBIB NIH HHS - U18 EB021780()
  • NIDDK NIH HHS - R01 DK070858()
  • NIDDK NIH HHS - R01 DK092456()
  • NIDDK NIH HHS - R01 DK098350()
  • NIDDK NIH HHS - R01 DK102551()
  • NIDDK NIH HHS - U01 DK103117()

Moxd1 Is a Marker for Sexual Dimorphism in the Medial Preoptic Area, Bed Nucleus of the Stria Terminalis and Medial Amygdala.

  • Tsuneoka Y
  • Front Neuroanat
  • 2017 Apr 11

Literature context:


Abstract:

The brain shows various sex differences in its structures. Various mammalian species exhibit sex differences in the sexually dimorphic nucleus of the preoptic area (SDN-POA) and parts of the extended amygdala such as the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr) and posterodorsal part of the medial amygdala (MePD). The SDN-POA and BNSTpr are male-biased sexually dimorphic nuclei, and characterized by the expression of calbindin D-28K (calbindin 1). However, calbindin-immunoreactive cells are not restricted to the SDN-POA, but widely distributed outside of the SDN-POA. To find genes that are more specific to sexually dimorphic nuclei, we selected candidate genes by searching the Allen brain atlas and examined the detailed expressions of the candidate genes using in situ hybridization. We found that the strong expression of monooxygenase DBH-like 1 (Moxd1) was restricted to the SDN-POA, BNSTpr and MePD. The numbers of Moxd1-positive cells in the SDN-POA, BNSTpr and MePD in male mice were larger than those in female mice. Most of the Moxd1-positive cells in the SDN-POA and BNSTpr expressed calbindin. Neonatal castration of male mice reduced the number of Moxd1-positive cells in the SDN-POA, whereas gonadectomy in adulthood did not change the expression of the Moxd1 gene in the SDN-POA in both sexes. These results suggest that the Moxd1 gene is a suitable marker for sexual dimorphic nuclei in the POA, BNST and amygdala, which enables us to manipulate sexually dimorphic neurons to examine their roles in sex-biased physiology and behaviors.

Tridimensional Visualization and Analysis of Early Human Development.

  • Belle M
  • Cell
  • 2017 Mar 23

Literature context:


Abstract:

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.

Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells.

  • Jiang T
  • Elife
  • 2017 Mar 7

Literature context:


Abstract:

The asymmetric location of stereociliary bundle (hair bundle) on the apical surface of mechanosensory hair cells (HCs) dictates the direction in which a given HC can respond to cues such as sound, head movements, and water pressure. Notably, vestibular sensory organs of the inner ear, the maculae, exhibit a line of polarity reversal (LPR) across which, hair bundles are polarized in a mirror-image pattern. Similarly, HCs in neuromasts of the zebrafish lateral line system are generated as pairs, and two sibling HCs develop opposite hair bundle orientations. Within these sensory organs, expression of the transcription factor Emx2 is restricted to only one side of the LPR in the maculae or one of the two sibling HCs in neuromasts. Emx2 mediates hair bundle polarity reversal in these restricted subsets of HCs and generates the mirror-image pattern of the sensory organs. Downstream effectors of Emx2 control bundle polarity cell-autonomously via heterotrimeric G proteins.

CREB Signaling Is Involved in Rett Syndrome Pathogenesis.

  • Bu Q
  • J. Neurosci.
  • 2017 Mar 29

Literature context:


Abstract:

Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene. To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation (MECP2T158M/T158M ), hESC line expressing no MECP2 (MECP2-KO), congenic pair of wild-type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function. At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated from MECP2T158M/T158M , MECP2-KO, and V247fs-MT stem cell lines. Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the female Mecp2 heterozygous mice rescued several behavioral defects. Together, our study establishes a robust in vitro platform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT.SIGNIFICANCE STATEMENT Our study establishes a robust human stem cell-based platform for consistent quantitative evaluation of genotype-dependent Rett syndrome (RTT) phenotypes at the cellular level. By providing the first evidence that enhancing cAMP response element binding protein signaling can alleviate RTT phenotypes both in vitro and in vivo, we reveal a previously unappreciated role of cAMP response element binding protein signaling in RTT pathogenesis, and identify a potential therapeutic target for RTT.