Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage

YesGenetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.Cancer Research UK. Grant Number: A15673. Medical Research Council. Grant Number: MC_PC_U127527200. National Centre for the Replacement Refinement and Reduction of Animals in Research. Grant Number: NC/K001612/1. North West Cancer Research Fund. Grant Number: CR1132. Medical Research Scotland. Grant Number: 436FR

The composition of the protosolar disk and the formation conditions for comets

Conditions in the protosolar nebula have left their mark in the composition of cometary volatiles, thought to be some of the most pristine material in the solar system. Cometary compositions represent the end point of processing that began in the parent molecular cloud core and continued through the collapse of that core to form the protosun and the solar nebula, and finally during the evolution of the solar nebula itself as the cometary bodies were accreting. Disentangling the effects of the various epochs on the final composition of a comet is complicated. But comets are not the only source of information about the solar nebula. Protostellar disks around young stars similar to the protosun provide a way of investigating the evolution of disks similar to the solar nebula while they are in the process of evolving to form their own solar systems. In this way we can learn about the physical and chemical conditions under which comets formed, and about the types of dynamical processing that shaped the solar system we see today. This paper summarizes some recent contributions to our understanding of both cometary volatiles and the composition, structure and evolution of protostellar disks.Comment: To appear in Space Science Reviews. The final publication is available at Springer via http://dx.doi.org/10.1007/s11214-015-0167-

Open Source ImmGen: network perspective on metabolic diversity among mononuclear phagocytes

We dissect metabolic variability of mononuclear phagocyte (MNP) subpopulations across different tissues through integrative analysis of three large scale datasets. Specifically, we introduce ImmGen MNP Open Source dataset that profiled 337 samples and extended previous ImmGen effort which included 202 samples of mononuclear phagocytes and their progenitors. Next, we analysed Tabula Muris Senis dataset to extract data for 51,364 myeloid cells from 18 tissues. Taken together, a compendium of data assembled in this work covers phagocytic populations found across 38 different tissues. To analyse common metabolic features, we developed novel network-based computational approach for unbiased identification of key metabolic subnetworks based on cellular transcriptional profiles in large-scale datasets. Using ImmGen MNP Open Source dataset as baseline, we define 9 metabolic subnetworks that encapsulate the metabolic differences within mononuclear phagocytes, and demonstrate that these features are robustly found across all three datasets, including lipid metabolism, cholesterol biosynthesis, glycolysis, and a set of fatty acid related metabolic pathways, as well as nucleotide and folate metabolism. We systematically define major features specific to macrophage and dendritic cell subpopulations. Among other things, we find that cholesterol synthesis appears particularly active within the migratory dendritic cells. We demonstrate that interference with this pathway through statins administration diminishes migratory capacity of the dendritic cells in vivo. This result demonstrates the power of our approach and highlights importance of metabolic diversity among mononuclear phagocytes

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages

Systemic Analysis of PPARy gamma in Mouse Macrophage Populations Reveals Marked Diversity in Expression with Critical Roles in Resolution of Inflammation and Airway Immunity

Although PPARγ has anti-inflammatory actions in macrophages, which macrophage populations express PPARγ in vivo and how it regulates tissue homeostasis in the steady-state and during inflammation remains unclear. We now show that lung and spleen macrophages selectively expressed PPARγ among resting tissue macrophages. In addition, Ly-6C(hi) monocytes recruited to an inflammatory site induced PPARγ as they differentiated to macrophages. When PPARγ was absent in Ly-6C(hi)-derived inflammatory macrophages, initiation of the inflammatory response was unaffected but full resolution of inflammation failed, leading to chronic leukocyte recruitment. Conversely, PPARγ activation favors resolution of inflammation in a macrophage PPARγ-dependent manner. In the steady state, PPARγ deficiency in red pulp macrophages did not induce overt inflammation in the spleen. By contrast, PPARγ deletion in lung macrophages induced mild pulmonary inflammation at the steady-state and surprisingly precipitated mortality upon infection with S. pneumoniae. This accelerated mortality was associated with impaired bacterial clearance and inability to sustain macrophages locally. Overall, we uncovered critical roles for macrophage PPARγ in promoting resolution of inflammation and maintaining functionality in lung macrophages where it plays a pivotal role in supporting pulmonary host defense. Additionally, this work identifies specific macrophage populations as potential targets for the anti-inflammatory actions of PPARγ agonists

Comparison of gene expression profiles between human and mouse monocyte subsets.

Blood of both humans and mice contains 2 main monocyte subsets. Here, we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation, some molecules were conversely expressed between the 2 species' subsets, including CD36, CD9, and TREM-1. Other differences included a prominent peroxisome proliferator-activated receptor gamma (PPARgamma) signature in mouse monocytes, which is absent in humans, and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus, whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized, important differences between the species deserve consideration when models of human disease are studied in mice