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Background GATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury. Methods To determine GATA3's role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous (+/-) knockout mice, as well as injured human and rodent kidneys. Results We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis. Conclusions These results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections.

Accurate control of gene expression in the right cell at the right moment is of fundamental importance to animal development and homeostasis. At the heart of gene regulation lie the enhancers, a class of gene regulatory elements that ensures precise spatiotemporal activation of gene transcription. Mammalian genomes are littered with enhancers, which are frequently organized in cooperative clusters such as locus control regions and superenhancers. Here, we discuss our current knowledge of enhancer biology, including an overview of the discovery of the various enhancer subsets and the mechanistic models used to explain their gene regulatory function.

Summary Transcription factors are key players in a broad range of cellular processes such as cell-fate decision. Understanding how they act to control these processes is of critical importance for therapy purposes. FLI-1 controls several hematopoietic lineage differentiation including megakaryopoiesis and erythropoiesis. Its aberrant expression is often observed in cancer and is associated with poor prognosis. We showed that FLI-1 interacts with the LDB1 complex, which also plays critical roles in erythropoiesis and megakaryopoiesis. In this study, we aimed to unravel how FLI-1 and the LDB1 complex act together in murine erythroleukemia cells and in megakaryocyte. Combining omics techniques, we show that FLI-1 enables the recruitment of the LDB1 complex to regulatory sequences of megakaryocytic genes and to enhancers. We show as well for the first time that FLI-1 is able to modulate the 3D chromatin organization by promoting chromatin looping between enhancers and promoters most likely through the LDB1 complex., Graphical abstract, Highlights • FLI-1 is important for the recruitment of the LDB1 complex • FLI-1 is important for chromatin looping • FLI-1 and the LDB1 complex co-regulate megakaryopoiesis, Molecular Biology; Chromosome Organization

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

International audience; The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies.

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we have studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), where neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential to control apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and identified Sox6 as Zeb2-dependent and potential downstream target gene. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ involves non-autonomous mechanisms as well. Additionally, we could demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative manners in early postnatal life. ispartof: Development vol:147 issue:10 ispartof: location:England status: published

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells.

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.

// Anne-Sophie Gallouet 1, 2, 3, 4, 5 , Federica Ferri 1, 2, 3, 4, 5, 6, * , Vanessa Petit 1, 2, 3, 4, 5, * , Aude Parcelier 1, 2, 3, 4, 5 , Daniel Lewandowski 1, 2, 3, 4, 5 , Nathalie Gault 1, 2, 3, 4, 5 , Vilma Barroca 1, 2, 3, 4, 5 , Stephanie Le Gras 6 , Eric Soler 1, 2, 3, 4, 7 , Frank Grosveld 7 , Irwin Davidson 6 , Paul-Henri Romeo 1, 2, 3, 4, 5 1 CEA/DRF/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France 2 Inserm U967, 92265 Fontenay-aux-Roses cedex, France 3 Universite Paris-Diderot, Paris 7, France 4 Universite Paris-Sud, Paris 11, France 5 Equipe labellisee Ligue contre le Cancer, France 6 Department of Functional Genomics and Cancer, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, C. U. Strasbourg, France 7 Department of Cell Biology, Erasmus Medical Center, DR Molenwaterplein 50, 3015GE, Rotterdam, The Netherlands * These authors have contributed equally to this work Correspondence to: Paul-Henri Romeo, email: paul-henri.romeo@cea.fr Keywords: TRIM33, PU.1, macrophage, inflammation, myeloid differentiation Received: June 07, 2016 Accepted: November 12, 2016 Published: December 10, 2016 ABSTRACT The tripartite motif (TRIM) family of proteins plays important roles in innate immunity and antimicrobial infection. None of these proteins has been shown to directly regulate transcription of genes in monocyte/macrophage except TRIM33 that we have recently shown to be a macrophage specific transcriptional inhibitor of Ifnb1 . Using ChIP-seq analyses, we now report that TRIM33 is bound to two fold more genes in immature than in mature myeloid cell lines. When located near the same genes, TRIM33 is bound to different sequences in the two cell lines suggesting a role of TRIM33 in both immature and mature myeloid cells. Accordingly, expression of TRIM33 in immature myeloid cells is necessary for efficient production of small peritoneal macrophages, monocytes and bone marrow derived macrophage (BMDM) and TRIM33 targets a subset of genes involved in the inflammatory response only in mature myeloid cells. Functionally, this targeting is associated with impaired repression of pathways regulating the late phases of lipopolysaccharide (LPS) activation of BMDM and a high sensitivity to LPS in vivo when the trim33 gene is inactivated in mature myeloid cells. These findings pinpoint TRIM33 as an important transcriptional actor of monocyte/macrophage mediated inflammation.

SummaryBrown adipose tissue (BAT) is an energy-dispensing thermogenic tissue that plays an important role in balancing energy metabolism. Lineage-tracing experiments indicate that brown adipocytes are derived from myogenic progenitors during embryonic development. However, adult skeletal muscle stem cells (satellite cells) have long been considered uniformly determined toward the myogenic lineage. Here, we report that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3′UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake, and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance, and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are downregulated in mice exposed to cold, resulting in de novo generation of satellite cell-derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity.

GABAergic interneurons mainly originate in the medial ganglionic eminence (MGE) of the embryonic ventral telencephalon (VT) and migrate tangentially to the cortex, guided by membrane-bound and secreted factors. We found that Sip1 (Zfhx1b, Zeb2), a transcription factor enriched in migrating cortical interneurons, is required for their proper differentiation and correct guidance. The majority of Sip1 knockout interneurons fail to migrate to the neocortex and stall in the VT. RNA sequencing reveals that Sip1 knockout interneurons do not acquire a fully mature cortical interneuron identity and contain increased levels of the repulsive receptor Unc5b. Focal electroporation of Unc5b-encoding vectors in the MGE of wild-type brain slices disturbs migration to the neocortex, whereas reducing Unc5b levels in Sip1 knockout slices and brains rescues the migration defect. Our results reveal that Sip1, through tuning of Unc5b levels, is essential for cortical interneuron guidance. ispartof: Neuron vol:77 issue:1 pages:70-82 ispartof: location:United States status: published

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure‐to‐function relationship. However, all 3C‐based methods rely on chemical cross‐linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

Alveolarization of the developing lung is an important step toward the switch from intrauterine life to breathing oxygen-rich air after birth. The distal airways structurally change to minimize the gas exchange path, and Type II pneumocytes increase the production of surfactants, which are required to reduce surface tension at the air-liquid interface in the alveolus. Hypoxia-inducible factor 2 alpha (Hif2 alpha) is an oxygen-regulated transcription factor expressed in endothelial and Type II cells, and its expression increases toward the end of gestation. We investigated the role of Hif2 alpha in Type II cells by conditionally expressing an oxygen-insensitive mutant of Hif2 alpha in airway epithelial cells during development. Newborn mice expressing the mutant Hif2 alpha were born alive but quickly succumbed to respiratory distress. Subsequent analysis of the lungs revealed dilated alveoli covered with enlarged, aberrant Type II cells and a diminished number of Type I cells. The Type II cells accumulated glycogen in part by increased glucose uptake via the up-regulation of the glucose transporter 1. Furthermore, the cells lacked two crucial enzymes involved in the metabolism of glycogen into surfactant lipids, lysophosphatidylcholine acyltransferase and ATP-binding cassette sub-family A member 3. We conclude that Hif2 alpha is a key regulator in alveolar maturation and the production of phospholipids by Type II cells.

Various factors play an essential role in patterning the digestive tract. During development, Sox2 and Cdx2 are exclusively expressed in the anterior and the posterior parts of the primitive gut, respectively. However, it is unclear whether these transcription factors influence each other in determining specification of the naïve gut endoderm. We therefore investigated whether Sox2 redirects the fate of the prospective intestinal part of the primitive gut. Ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastric-like phenotype. Sox2 activated the foregut transcriptional program, in spite of sustained co-expression of endogenous Cdx2. However, binding of Cdx2 to its genomic targets and thus its transcriptional activity was strongly reduced. Recent findings indicate that endodermal Cdx2 is required to initiate the intestinal program and to suppress anterior cell fate. Our findings suggest that reduced Cdx2 expression by itself is not sufficient to cause anteriorization, but that Sox2 expression is also required. Moreover, it indicates that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut and that Sox2 may overrule the initial patterning of the primitive gut, emphasizing the plasticity of the primitive gut.

The HMG-box transcription factor Sox2 plays a role throughout neurogenesis and also acts at other stages of development, as illustrated by the multiple organs affected in the anophthalmia syndrome caused by SOX2 mutations. Here we combined proteomic and genomic approaches to characterize gene regulation by Sox2 in neural stem cells. Chd7, a chromatin remodeling ATPase associated with CHARGE syndrome, was identified as a Sox2 transcriptional cofactor. Sox2 and Chd7 physically interact, have overlapping genome-wide binding sites and regulate a set of common target genes including Jag1, Gli3 and Mycn, genes mutated in Alagille, Pallister-Hall and Feingold syndromes, which show malformations also associated with SOX2 anophthalmia syndrome or CHARGE syndrome. Regulation of disease-associated genes by a Sox2-Chd7 complex provides a plausible explanation for several malformations associated with SOX2 anophthalmia syndrome or CHARGE syndrome. Indeed, we found that Chd7-haploinsufficient embryos showed severely reduced expression of Jag1 in the developing inner ear. © 2011 Nature America, Inc. All rights reserved.

Panton–Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus -related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti–LukS-PV HCAb, three anti–LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti–LukS-PV HCAb also binds to γ-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies.

The transcription factor Gata3 is essential for the development of sympathetic neurons and adrenal chromaffin cells. As Gata3 expression is maintained up to the adult stage, we addressed its function in differentiated sympathoadrenal cells at embryonic and adult stages by conditional Gata3 elimination. Inactivation of Gata3 in embryonicDBH-expressing neurons elicits a strong reduction in neuron numbers due to apoptotic cell death and reduced proliferation. No selective effect on noradrenergic gene expression (THandDBH) was observed. Interestingly, Gata3 elimination inDBH-expressing neurons of adult animals also results in a virtually complete loss of sympathetic neurons. In the Gata3-deficient population, the expression of anti-apoptotic genes (Bcl-2,Bcl-xL, andNFκB) is diminished, whereas the expression of pro-apoptotic genes (Bik,Bok, andBmf) was increased. The expression of noradrenergic genes (THandDBH) is not affected. These results demonstrate that Gata3 is continuously required for maintaining survival but not differentiation in the sympathetic neuron lineage up to mature neurons of adult animals.

SRY and other Sox-type transcription factors are important developmental regulators with various implications in human disease. In this study, we identified Exp4 (exportin 4) as an interaction partner of Sox2 in mouse embryonic stem cells and neural progenitors. We show that, besides its established function in nuclear export, Exp4 acts as a bona fide nuclear import receptor for Sox2 and SRY. Thus, Exp4 is an example of a nuclear transport receptor carrying distinct cargoes into different directions. In contrast to a published study, we observed that the import activity of Imp-α (importin-a) isoforms toward Sox2 is negligible. Instead, we found that Imp9 and the Imp-β/7 heterodimer mediate nuclear import of Sox2 in parallel to Exp4. Import signals for the three pathways overlap and include conserved residues in the Sox2 high-mobility group (HMG) box domain that are also critical for DNA binding. This suggests that nuclear import of Sox proteins is facilitated by several parallel import pathways.

Phospholipid transfer protein (PLTP) is a multifunctional protein synthesized by various cell types and secreted into the plasma. Plasma PLTP is able to transfer phospholipids between lipoproteins and modulate HDL particles. Mice with overexpression of human PLTP have an increased ability to generate pre beta-HDL, reduced total HDL levels and an increased susceptibility to atherosclerosis. As the macrophage is a key component of the atherosclerotic lesion and an important site of PLTP expression, we investigated the role of systemic and peripheral PLTP in macrophage cholesterol efflux and reverse cholesterol transport (RCT) in vivo. We used an assay in which H-3-labelled cholesterol-loaded macrophages were injected intraperitoneally into recipient mice, and radioactivity was quantified in plasma, liver and faeces. Firstly, wild type macrophages were injected into wild type, PLTP transgenic (PLTPtg) and apoAI transgenic (apoAItg) mice. While plasma H-3-tracer levels in apoAItg mice were increased compared with wild type mice, they were reduced in PLTPtg mice. Moreover, overexpression of PLTP significantly decreased faecal H-3-tracer levels compared with wild type and apoAItg mice. Secondly, wild type mice were injected with peritoneal macrophages derived from PLTPtg or wild type mice. No significant difference in the amount of H-3-tracer in plasma, liver or faeces was found between the two groups of mice. Our findings demonstrate that macrophage cholesterol efflux and RCT to faeces is impaired in PLTP transgenic mice, and that elevation of macrophage-PLTP does not affect RCT, indicating that higher systemic PLTP levels may promote atherosclerosis development by decreasing the rate of macrophage RCT. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Plasma phospholipid transfer protein ( PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride ( TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene ( mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.-Samyn, H., M. Moerland, T. van Gent, R. van Haperen, J. Metso, F. Grosveld, M. Jauhiainen, A. van Tol, and R. de Crom. Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: a mutation-inactivation study. J. Lipid Res. 2008. 49: 2504-2512.

In mammalian female cells, one X chromosome is inactivated to prevent a dose difference in the expression of X-encoded proteins between males and females. Xist RNA, required for X chromosome inactivation, is transcribed from the future inactivated X chromosome (Xi), where it spreads in cis, to initiate silencing. We have analyzed Xist RNA transcription and localization throughout the cell cycle. It was found that Xist transcription is constant and that the mature RNA remains attached to the Xi throughout mitosis. Diploid and tetraploid cell lines with an MS2-tagged Xist gene were used to investigate spreading of Xist. Most XXXX(MS2) tetraploid mouse embryonic stem (ES) cells inactivate the X(MS2) chromosome and one other X chromosome. Analysis of cells with two Xi's indicates that Xist RNA is retained by the Xi of its origin and does not spread in trans. Also, in XX(MS2) diploid mouse ES cells with an autosomal Xist transgene, there is no trans exchange of Xist RNA from the Xi to the autosome. We propose that Xist RNA does not dissociate from the Xi of its origin, which precludes a model of diffusion-mediated trans spreading of Xist RNA.

Objective— A transgenic mouse model was generated that allows conditional expression of human PLTP, based on the tetracycline-responsive gene system, to study the effects of an acute increase in plasma PLTP activity as may occur in inflammation. Methods and Results— The effects of an acute elevation of plasma PLTP activity on the metabolism of apolipoprotein B–containing lipoproteins and on diet-induced pre-existing atherosclerosis were determined in mice displaying a humanized lipoprotein profile (low-density lipoprotein receptor knockout background). Induced expression of PLTP strongly increases plasma VLDL levels in LDL receptor knockout mice, whereas VLDL secretion is not affected. The elevation in plasma triglyceride levels is explained by a PLTP-dependent inhibition of VLDL catabolism, which is caused, at least partly, by a decreased lipoprotein lipase activity. Together with the decreased plasma HDL levels, the acutely increased PLTP expression results in a highly atherogenic lipoprotein profile. Induction of PLTP expression leads to a further increase in size of pre-existing atherosclerotic lesions, even on a chow diet. In addition, the lesions contain more macrophages and less collagen relative to controls, suggesting a less stable lesion phenotype. Conclusion— In conclusion, acute elevation of PLTP activity destabilizes atherosclerotic lesions and aggravates pre-existing atherosclerosis.

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

The transcription factor Gata3 is crucially involved in epidermis and hair follicle differentiation. Yet, little is known about how Gata3 co-ordinates stem cell lineage determination in skin, what pathways are involved and how Gata3 differentially regulates distinct cell populations within the hair follicle. Here, we describe a conditional Gata3-/- mouse(K14-Gata3-/-) in which Gata3 is specifically deleted in epidermis and hair follicles. K14-Gata3-/- mice show aberrant postnatal growth and development, delayed hair growth and maintenance, abnormal hair follicle organization and irregular pigmentation. After the first hair cycle, the germinative layer surrounding the dermal papilla was not restored; instead, proliferation was pronounced in basal epidermal cells. Transcriptome analysis of laser-dissected K14-Gata3-/- hair follicles revealed mitosis, epithelial differentiation and the Notch, Wnt and BMP signaling pathways to be significantly overrepresented. Elucidation of these pathways at the RNA and protein levels and physiologic endpoints suggests that Gata3 integrates diverse signaling networks to regulate the balance between hair follicle and epidermal cell fates.

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time, and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies, however, on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with (111)In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast-growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation than slow-growing xenografts. Additionally, several other parameters known to influence the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of infiltrating macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation, and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However, liposomal uptake was significantly correlated with tumor growth, with fast-growing tumors showing a higher uptake, although no biological determinants could be elucidated to explain this correlation.

The transcription factor Gata-3 is a defining marker of the 'luminal' subtypes of breast cancer. To gain insight into the role of Gata-3 in breast epithelial development and oncogenesis, we have explored its normal function within the mammary gland by conditionally deleting Gata-3 at different stages of development. We report that Gata-3 has essential roles in the morphogenesis of the mammary gland in both the embryo and adult. Through the discovery of a novel marker (beta3-integrin) of luminal progenitor cells and their purification, we demonstrate that Gata-3 deficiency leads to an expansion of luminal progenitors and a concomitant block in differentiation. Remarkably, introduction of Gata-3 into a stem cell-enriched population induced maturation along the alveolar luminal lineage. These studies provide evidence for the existence of an epithelial hierarchy within the mammary gland and establish Gata-3 as a critical regulator of luminal differentiation.

Background— Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies. Methods and Results— We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38±0.68 versus 0.22±0.04); contained fewer smooth muscle cells (1.9±1.6% versus 26.3±9.7%), less collagen (15.3±1.0% versus 22.2±1.0%), and more lipids (15.8±0.9% versus 10.2±0.5%); and showed more outward vascular remodeling (214±19% versus 117±9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II–induced intraplaque hemorrhages occurred in the lowered shear stress regions only. Conclusions— Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.

We have generated transgenic mice containing hybrid llama/human antibody loci that contain two llama variable regions and the human D, J, and Cμ and/or Cγ constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-chain-only antibodies (HCAb) are expressed at high levels, provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling. Antigen-specific heavy-chain-only IgM or IgGs are produced upon immunization. The IgG is dimeric, whereas IgM is multimeric. The chimeric HCAb loci are subject to allelic exclusion, but several copies of the transgenic locus can be rearranged and expressed successfully on the same allele in the same cell. Such cells are not subject to negative selection. The mice produce a full antibody repertoire and provide a previously undescribed avenue to produce specific human HCAb in the future.

CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS. LL5beta and ELKS form a complex that colocalizes with CLASPs at the cortex of HeLa cells as well as at the leading edge of motile fibroblasts. LL5beta is required for cortical CLASP accumulation and microtubule stabilization in HeLa cells, while ELKS plays an accessory role in these processes. LL5beta is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein, and its recruitment to the cell cortex is influenced by PI3 kinase activity but does not require intact microtubules. Cortical clusters of LL5beta and ELKS do not overlap with focal adhesions but often form in their vicinity and can affect their size. We propose that LL5beta and ELKS can form a PIP3-regulated cortical platform to which CLASPs attach distal microtubule ends.

Contains fulltext : 50157.pdf (Publisher’s version ) (Closed access) In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.

Objective To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. Methods Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. Results We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. Conclusions The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.

Although several molecular players have been described that play a role during the early phases of lung development, it is still unknown how the vasculature develops in relation to the airways. Two opposing models describe development of lung vasculature: one suggests that both vasculogenesis and angiogenesis are involved, whereas the second describes vasculogenesis as the primary mechanism. Therefore, we examined the development of the murine pulmonary vasculature through a morphological analysis from the onset of lung development [9.5 days postcoital (dpc)] until the pseudoglandular stage (13.5 dpc). We analyzed fetal lungs of Tie2-LacZ transgenic mice as well as serial sections of wild-type lungs stained with endothelial-specific antibodies (Flk-1, Fli-1, and PECAM-1). Embryos were processed with intact blood circulation to maintain the integrity of the vasculature; hence individual vessels could be identified with accuracy through serial section analysis. Furthermore, circulating primitive erythrocytes, formed exclusively by the blood islands in the yolk sac, are trapped in vessels during fixation, which proves the connection with the embryonic circulation. We report that from the first morphological sign of lung development, a clear vascular network exists that is in contact with the embryonic circulation. We propose distal angiogenesis as a new concept for early pulmonary vascular morphogenesis. In this model, capillary networks surround the terminal buds and expand by formation of new capillaries from preexisting vessels as the lung bud grows. The fact that at an early embryonic stage a complete vascular network exists may be important for the general understanding of embryonic development.

International audience; CLIP-170 is a microtubule "plus-end-tracking protein" implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. Residual CLIP-170 is detected in lungs and embryos of homozygous CLIP-170 knockout mice, but not in other tissues and cell types, indicating that we have generated a hypomorphic mutant. Homozygous CLIP-170 knockout mice are viable and appear normal. However, male knockout mice are subfertile and produce sperm with abnormal heads. Using the knock-in mice, we followed GFP-CLIP-170 expression and behavior in dissected, live testis tubules. We detect plus-end-tracking GFP-CLIP-170 in spermatogonia. As spermatogenesis proceeds, GFP-CLIP-170 expression increases and the fusion protein strongly marks syncytia of differentiated spermatogonia and early prophase spermatocytes. Subsequently GFP-CLIP-170 levels drop, but during spermiogenesis (post-meiotic development), GFP-CLIP-170 accumulates again and is present on spermatid manchettes and centrosomes. Bleaching studies show that, as spermatogenesis progresses, GFP-CLIP-170 converts from a mobile plus-end-tracking protein to a relatively immobile protein. We propose that CLIP-170 has a structural function in the male germline, in particular in spermatid differentiation and sperm head shaping. Supplemental material is available at http://www.genesdev.org.

Mice homozygous for the autosomal recessive mutation claw paw (clp) are characterized by limb posture abnormalities and congenital hypomyelination, with delayed onset of myelination of the peripheral nervous system but not the central nervous system. Although this combination of limb and peripheral nerve abnormalities in clp/clp mice might suggest a common neurogenic origin of the syndrome, it is not clear whether the clp gene acts primarily in the neurone, the Schwann cell or both. In the work described here, we address this question of cell autonomy of the clp mutation through reciprocal nerve grafting experiments between wild-type and clp/clp animals. Our results demonstrate that the clp mutation affects the Schwann cell compartment and possibly also the neuronal compartment. These data suggest that the clp gene product is expressed in Schwann cells as well as neurones and is likely to be involved in direct axon–Schwann cell interactions. Within the Schwann cell, clp affects a myelin-related signaling pathway that regulates periaxin and Krox-20 expression, but not Oct-6.

As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/- mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre-B-cell proliferation and differentiation into immunoglobulin M-positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/- mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the null mutants precludes the analysis of adult hematopoiesis in Sp3-/- mice. We therefore investigated the ability of E12.5 Sp3-/- liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/- cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/- cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.

The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and thatBrn-2gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of theOct-6Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of bothBrn-2andOct-6results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells.

International audience; The GATA-3 transcription factor has a determinant role in T cell specification and is an essential mediator of T helper 2-type polarized immune responses. While both committed NK precursors and mature NK cells express GATA-3, a role of this transcription factor in murine NK cell differentiation is not known. We found that NK cells, in contrast to T cells, can be generated in the absence of GATA-3. However, while GATA-3 antagonizes IFN-gamma production in differentiating T cells, GATA-3-deficient NK cells paradoxically produced less IFN-gamma compared to control NK cells and failed to provide early protection in vivo against infection with Listeria monocytogenes. Surprisingly, GATA-3 was essential for NK cell homing to the liver. Our results suggest that GATA-3 promotes NK cell maturation and acts in this lineage to specify distinct effector phenotypes.