Publications by authors named "Ian J Jackson"

80 Publications

Genetic background modifies vulnerability to glaucoma-related phenotypes in mutant mice.

Dis Model Mech 2021 Feb 19;14(2). Epub 2021 Feb 19.

Howard Hughes Medical Institute, Department of Ophthalmology, Columbia University Medical Center, and Zuckerman Mind Brain Behavior Institute, New York, NY 10027, USA

Variants in the LIM homeobox transcription factor 1-beta () gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the (also known as ) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA- /J (C3H) and DBA/2J- (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to -induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating -induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments.
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http://dx.doi.org/10.1242/dmm.046953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903917PMC
February 2021

Developmental programming: Prenatal testosterone excess disrupts pancreatic islet developmental trajectory in female sheep.

Mol Cell Endocrinol 2020 12 26;518:110950. Epub 2020 Jul 26.

Department of Pediatrics, University of Michigan, Ann Arbor, MI, 48109, USA. Electronic address:

Prenatal testosterone (T)- treated female sheep manifest juvenile insulin resistance, post-pubertal increase in insulin sensitivity and return to insulin resistance during adulthood. Since compensatory hyperinsulinemia is associated with insulin resistance, altered pancreatic islet ontogeny may contribute towards metabolic defects. To test this, pregnant sheep were treated with or without T propionate from days 30-90 of gestation and pancreas collected from female fetuses at gestational day 90 and female offspring at 21 months-of-age. Uterine (maternal) and umbilical (fetal) arterial blood insulin/glucose ratios were determined at gestational day 90. The morphological and functional changes in pancreatic islet were assessed through detection of 1) islet hormones (insulin, glucagon) and apoptotic beta cells at fetal day 90 and 2) islet hormones (insulin, glucagon and somatostatin), and pancreatic lipid and collagen accumulation in adults. At gestational day 90, T-treatment led to maternal but not fetal hyperinsulinemia, decrease in pancreatic/fetal weight ratio and alpha cells, and a trend for increase in beta cell apoptosis in fetal pancreas. Adult prenatal T-treated female sheep manifested 1) significant increase in beta cell size and a tendency for increase in insulin and somatostatin stained area and proportion of beta cells in the islet; and 2) significant increase in pancreatic islet collagen and a tendency towards increased lipid accumulation. Gestational T-treatment induced changes in pancreatic islet endocrine cells during both fetal and adult ages track the trajectory of hyperinsulinemic status with the increase in adult pancreatic collagen accumulation indicative of impending beta cell failure with chronic insulin resistance.
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http://dx.doi.org/10.1016/j.mce.2020.110950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609617PMC
December 2020

The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification.

PLoS Genet 2020 04 1;16(4):e1008583. Epub 2020 Apr 1.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.
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http://dx.doi.org/10.1371/journal.pgen.1008583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153906PMC
April 2020

Fam151b, the mouse homologue of C.elegans menorin gene, is essential for retinal function.

Sci Rep 2020 01 16;10(1):437. Epub 2020 Jan 16.

MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Fam151b is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching. The International Mouse Phenotyping Consortium (IMPC) aims to knock out every gene in the mouse and comprehensively phenotype the mutant animals. This project identified Fam151b homozygous knock-out mice as having retinal degeneration. We show they have no photoreceptor function from eye opening, as demonstrated by a lack of electroretinograph (ERG) response. Histological analysis shows that during development of the eye the correct number of cells are produced and that the layers of the retina differentiate normally. However, after eye opening at P14, Fam151b mutant eyes exhibit signs of retinal stress and rapidly lose photoreceptor cells. We have mutated the second mammalian menorin homologue, Fam151a, and homozygous mutant mice have no discernible phenotype. Sequence analysis indicates that the FAM151 proteins are members of the PLC-like phosphodiesterase superfamily. However, the substrates and function of the proteins remains unknown.
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http://dx.doi.org/10.1038/s41598-019-57398-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965129PMC
January 2020

Missense Mutations in the Human Nanophthalmos Gene TMEM98 Cause Retinal Defects in the Mouse.

Invest Ophthalmol Vis Sci 2019 07;60(8):2875-2887

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.

Purpose: We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs.

Methods: The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes.

Results: An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions.

Conclusions: Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.
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http://dx.doi.org/10.1167/iovs.18-25954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986908PMC
July 2019

Genome-wide study of hair colour in UK Biobank explains most of the SNP heritability.

Nat Commun 2018 12 10;9(1):5271. Epub 2018 Dec 10.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.

Natural hair colour within European populations is a complex genetic trait. Previous work has established that MC1R variants are the principal genetic cause of red hair colour, but with variable penetrance. Here, we have extensively mapped the genes responsible for hair colour in the white, British ancestry, participants in UK Biobank. MC1R only explains 73% of the SNP heritability for red hair in UK Biobank, and in fact most individuals with two MC1R variants have blonde or light brown hair. We identify other genes contributing to red hair, the combined effect of which accounts for ~90% of the SNP heritability. Blonde hair is associated with over 200 genetic variants and we find a continuum from black through dark and light brown to blonde and account for 73% of the SNP heritability of blonde hair. Many of the associated genes are involved in hair growth or texture, emphasising the cellular connections between keratinocytes and melanocytes in the determination of hair colour.
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http://dx.doi.org/10.1038/s41467-018-07691-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288091PMC
December 2018

Mouse mutations cause retinal degeneration and reduced mitochondrial function.

Dis Model Mech 2018 12 18;11(12). Epub 2018 Dec 18.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK

Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one β and one γ subunit. Loss-of-function and missense mutations in both and have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months, but no other tissues appear to be affected. We produced a knockout of and found that homozygous mice do not survive past early embryogenesis. compound heterozygous mutants exhibit a more severe retinal degeneration compared with homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both and cells. Loss-of-function mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in mutant mice.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.036426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307916PMC
December 2018

A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice.

Dev Cell 2018 11;47(4):509-523.e5

Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Bailrigg, Furness Building, Lancaster LA1 4YG, UK. Electronic address:

The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease.
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http://dx.doi.org/10.1016/j.devcel.2018.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251972PMC
November 2018

Cell signalling: Red alert about lipid's role in skin cancer.

Nature 2017 09 6;549(7672):337-339. Epub 2017 Sep 6.

MRC Human Genetics Unit, University of Edinburgh, Edinburgh EH4 2XU, UK.

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http://dx.doi.org/10.1038/nature23550DOI Listing
September 2017

PLAA Mutations Cause a Lethal Infantile Epileptic Encephalopathy by Disrupting Ubiquitin-Mediated Endolysosomal Degradation of Synaptic Proteins.

Am J Hum Genet 2017 May 13;100(5):706-724. Epub 2017 Apr 13.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK. Electronic address:

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
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http://dx.doi.org/10.1016/j.ajhg.2017.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420347PMC
May 2017

KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability.

J Cell Biol 2017 04 28;216(4):999-1013. Epub 2017 Feb 28.

Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU Scotland, UK

Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive "actin gate" that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics.
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http://dx.doi.org/10.1083/jcb.201607032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379941PMC
April 2017

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Nat Commun 2016 08 18;7:12444. Epub 2016 Aug 18.

MRC Harwell Institute, Mammalian Genetics Unit, Harwell Campus, Oxfordshire OX11 0RD, UK.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.
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http://dx.doi.org/10.1038/ncomms12444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992138PMC
August 2016

Reconciling diverse mammalian pigmentation patterns with a fundamental mathematical model.

Nat Commun 2016 Jan 6;7:10288. Epub 2016 Jan 6.

Centre for Mathematical Biology, Department of Mathematical Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK.

Bands of colour extending laterally from the dorsal to ventral trunk are a common feature of mouse chimeras. These stripes were originally taken as evidence of the directed dorsoventral migration of melanoblasts (the embryonic precursors of melanocytes) as they colonize the developing skin. Depigmented 'belly spots' in mice with mutations in the receptor tyrosine kinase Kit are thought to represent a failure of this colonization, either due to impaired migration or proliferation. Tracing of single melanoblast clones, however, has revealed a diffuse distribution with high levels of axial mixing--hard to reconcile with directed migration. Here we construct an agent-based stochastic model calibrated by experimental measurements to investigate the formation of diffuse clones, chimeric stripes and belly spots. Our observations indicate that melanoblast colonization likely proceeds through a process of undirected migration, proliferation and tissue expansion, and that reduced proliferation is the cause of the belly spots in Kit mutants.
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http://dx.doi.org/10.1038/ncomms10288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729835PMC
January 2016

The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

Dis Model Mech 2015 Dec 5;8(12):1555-68. Epub 2015 Nov 5.

MRC Mammalian Genetics Unit, MRC Harwell, Oxford, OX11 0RD, UK

Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.
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http://dx.doi.org/10.1242/dmm.023176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728324PMC
December 2015

Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation.

Open Biol 2015 Jun;5(6):150047

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.

RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary 'RAB3GAP' complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.
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http://dx.doi.org/10.1098/rsob.150047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632505PMC
June 2015

Maintenance of distinct melanocyte populations in the interfollicular epidermis.

Pigment Cell Melanoma Res 2015 Jul 30;28(4):476-80. Epub 2015 Apr 30.

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, UK.

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.
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http://dx.doi.org/10.1111/pcmr.12375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973853PMC
July 2015

The melanocyte lineage in development and disease.

Development 2015 Apr;142(7):1387

MRC Human Genetics Unit and University of Edinburgh Cancer Research UK Cancer Centre, MRC Institute for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.

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http://dx.doi.org/10.1242/dev.123729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378253PMC
April 2015

Mouse slc9a8 mutants exhibit retinal defects due to retinal pigmented epithelium dysfunction.

Invest Ophthalmol Vis Sci 2015 May;56(5):3015-26

MRC Human Genetics Unit, MRC Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom 4Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.

Purpose: As part of a large scale systematic screen to determine the effects of gene knockout mutations in mice, a retinal phenotype was found in mice lacking the Slc9a8 gene, encoding the sodium/hydrogen ion exchange protein NHE8. We aimed to characterize the mutant phenotype and the role of sodium/hydrogen ion exchange in retinal function.

Methods: Detailed histology characterized the pathological consequences of Slc9a8 mutation, and retinal function was assessed by electroretinography (ERG). A conditional allele was used to identify the cells in which NHE8 function is critical for retinal function, and mutant cells analyzed for the effect of the mutation on endosomes.

Results: Histology of mutant retinas reveals a separation of photoreceptors from the RPE and infiltration by macrophages. There is a small reduction in photoreceptor length and a mislocalization of visual pigments. The ERG testing reveals a deficit in rod and cone pathway function. The RPE shows abnormal morphology, and mutation of Slc9a8 in only RPE cells recapitulates the mutant phenotype. The NHE8 protein localizes to endosomes, and mutant cells have much smaller recycling endosomes.

Conclusions: The NHE8 protein is required in the RPE to maintain correct regulation of endosomal volume and/or pH which is essential for the cellular integrity and subsequent function of RPE.
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http://dx.doi.org/10.1167/iovs.14-15735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538965PMC
May 2015

The melanocyte lineage in development and disease.

Development 2015 Feb;142(4):620-32

MRC Human Genetics Unit and Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK.

Melanocyte development provides an excellent model for studying more complex developmental processes. Melanocytes have an apparently simple aetiology, differentiating from the neural crest and migrating through the developing embryo to specific locations within the skin and hair follicles, and to other sites in the body. The study of pigmentation mutations in the mouse provided the initial key to identifying the genes and proteins involved in melanocyte development. In addition, work on chicken has provided important embryological and molecular insights, whereas studies in zebrafish have allowed live imaging as well as genetic and transgenic approaches. This cross-species approach is powerful and, as we review here, has resulted in a detailed understanding of melanocyte development and differentiation, melanocyte stem cells and the role of the melanocyte lineage in diseases such as melanoma.
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http://dx.doi.org/10.1242/dev.106567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325379PMC
February 2015

Spinster homolog 2 (spns2) deficiency causes early onset progressive hearing loss.

PLoS Genet 2014 Oct 30;10(10):e1004688. Epub 2014 Oct 30.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Wolfson Centre for Age-Related Diseases, King's College London, London, United Kingdom.

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.
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http://dx.doi.org/10.1371/journal.pgen.1004688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214598PMC
October 2014

HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

PLoS Genet 2014 Sep 18;10(9):e1004577. Epub 2014 Sep 18.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.
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http://dx.doi.org/10.1371/journal.pgen.1004577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168999PMC
September 2014

Genetic background influences age-related decline in visual and nonvisual retinal responses, circadian rhythms, and sleep.

Neurobiol Aging 2015 Jan 2;36(1):380-93. Epub 2014 Aug 2.

MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK. Electronic address:

The circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep.
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http://dx.doi.org/10.1016/j.neurobiolaging.2014.07.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270439PMC
January 2015

Loss of ALDH18A1 function is associated with a cellular lipid droplet phenotype suggesting a link between autosomal recessive cutis laxa type 3A and Warburg Micro syndrome.

Mol Genet Genomic Med 2014 Jul 11;2(4):319-25. Epub 2014 Mar 11.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh Edinburgh, UK.

Autosomal recessive cutis laxa type 3A is caused by mutations in ALDH18A1, a gene encoding the mitochondrial enzyme Δ(1)-pyrroline-5-carboxylate synthase (P5CS). It is a rare disorder with only six pathogenic mutations and 10 affected individuals from five families previously described in the literature. Here we report the identification of novel compound heterozygous missense mutations in two affected siblings from a Lebanese family by whole-exome sequencing. The mutations alter a conserved C-terminal domain of the encoded protein and reduce protein stability as determined through Western blot analysis of patient fibroblasts. Patient fibroblasts exhibit a lipid droplet phenotype similar to that recently reported in Warburg Micro syndrome, a disorder with similar features but hitherto unrelated cellular etiology.
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http://dx.doi.org/10.1002/mgg3.70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113272PMC
July 2014

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

J Vis Exp 2014 May 19(87). Epub 2014 May 19.

MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh;

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.
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http://dx.doi.org/10.3791/51352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4193345PMC
May 2014

A dominant-negative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LDB1-mediated dimerization [corrected].

PLoS Genet 2014 May 8;10(5):e1004359. Epub 2014 May 8.

MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, United Kingdom; The Roslin Institute, University of Edinburgh, Easter Bush, United Kingdom.

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.
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http://dx.doi.org/10.1371/journal.pgen.1004359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014447PMC
May 2014

A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

Dis Model Mech 2014 Jun 24;7(6):711-22. Epub 2014 Apr 24.

MRC Human Genetics Unit, Institute for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK. The Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK.

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.
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http://dx.doi.org/10.1242/dmm.015222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036478PMC
June 2014

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis.

Mol Biol Cell 2014 Apr 19;25(8):1216-33. Epub 2014 Feb 19.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1HH, United Kingdom Biomedical Sciences Research Complex Mass Spectrometry and Proteomics Facility, University of St. Andrews, St. Andrews, Fife KY16 9ST, United Kingdom.

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.
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http://dx.doi.org/10.1091/mbc.E13-08-0471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982988PMC
April 2014

Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes.

PLoS Genet 2013 26;9(12):e1003928. Epub 2013 Dec 26.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.
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http://dx.doi.org/10.1371/journal.pgen.1003928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887133PMC
August 2014

A trans-acting protein effect causes severe eye malformation in the Mp mouse.

PLoS Genet 2013 12;9(12):e1003998. Epub 2013 Dec 12.

The MRC Human Genetics Unit, MRC Institute of Genetic and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.
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http://dx.doi.org/10.1371/journal.pgen.1003998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861116PMC
August 2014