Biology subjects

Murray, S. A.

Publications and source records attributed to Murray, S. A..

32 records · Page 2Linked to original sources

bioRxiv2022

OPA1 Downregulation in Skeletal Muscle Induces MERC formation in an ATF4-Dependent Manner

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

biophysics↗

bioRxiv2022

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo. SIGNIFICANCE STATEMENTModifying the DNA of cells in the brain could present opportunities for new treatments of neurological diseases. In this report, we describe a nanocapsule system designed to deliver the elements needed to modify the DNA of brain cells, also known as genome editing. These nanocapsules are created by chemically encapsulating the genome editing components, such that the nanocapsules are stable when prepared and biodegradable to release their payload upon entering cells. When injected into the mouse brain, our research shows that the nanocapsules lead to safe and efficient editing of DNA in neurons.

neuroscience↗

bioRxiv2022

MICOS Complex and Mitochondria Morphology Changes Across Aging in Cardiac Muscle

ABSTRACTWith sparse treatment options, cardiac disease remains a significant cause of death among humans. As a person ages, mitochondria break down and the heart becomes less efficient. Heart failure is linked to many mitochondria-associated processes, including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress. The roles of key mitochondrial complexes that dictate the ultrastructure, such as the mitochondrial contact site and cristae organizing system (MICOS), in aging cardiac muscle are poorly understood. To better understand the cause of age-related alteration in mitochondrial structure in cardiac muscle, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the 3D networks in cardiac muscle samples of male mice at aging intervals of 3 months, 1 year, and 2 years. Here, we present the loss of cristae morphology, the inner folds of the mitochondria, across age. In conjunction with this, the 3D volume of mitochondria decreased. These findings mimicked observed phenotypes in murine cardiac fibroblasts with CRISPR/Cas9 knockout of Mitofilin, Chchd3, Chchd6 (some members of the MICOS complex), and Opa1, which showed poorer oxidative consumption rate and mitochondria with decreased mitochondrial length and volume. In combination, these data show the need to explore if loss of the MICOS complex in the heart may be involved in age-associated mitochondrial and cristae structural changes.

biophysics↗

bioRxiv2022

Profiling Mouse Brown and White Adipocytes to Identify Metabolically Relevant Small ORFs and Functional Microproteins

The absence of thousands of recently annotated small open reading frame (smORF)-encoded peptides and small proteins (microproteins) from databases has precluded their analysis in metabolism and metabolic disease. Given the outsized importance of small proteins and peptides such as insulin, leptin, amylin, glucagon, and glucagon-like peptide-1 (GLP-1) in metabolism, microproteins are a potentially rich source of uncharacterized metabolic regulators. Here, we annotate smORFs in primary differentiated brown, white, and beige mouse adipose cells. Ribosome profiling (Ribo-Seq) detected a total of 3,877 unannotated smORFs. Analysis of RNA-Seq datasets revealed diet-regulated smORF expression in adipose tissues, and validated the adipose translation of the feeding-neuron marker gene Gm8773. Gm8773 encodes the mouse homolog of FAM237B, a neurosecretory protein that stimulates food intake and promotes weight gain in chickens. Testing of recombinant mFAM237B produced similar orexigenic activity in mice further supporting a role for FAM237B as a metabolic regulator and potentially part of the brain-adipose axis. Furthermore, we demonstrated that data independent acquisition mass spectrometry (DIA-MS) proteomics can provide a sensitive, flexible, and quantitative platform for identifying microproteins by mass spectrometry. Using this system led to the detection of 58 microproteins from cell culture and an additional 33 from mouse plasma. The proteomics data established the anti-inflammatory microprotein AW112010 as a circulating factor, and found that plasma levels of a microprotein translated from a FRS2 uORF is elevated in older obese mice. Together, the data highlight the value of this database in examining understudied smORFs and microproteins in metabolic research and identifying additional regulators of metabolism.

systems biology↗

bioRxiv2021

The Role of Mentoring in Promoting Diversity, Equity, and Inclusion in STEM Education and Research

Mentoring success is derived from active and respectful listening and the willingness to learn and accept opportunities for personal growth. Mentoring shapes every trainee and their career path in science, technology, engineering, and mathematics (STEM). Productive mentoring relationships cultivate rapport, stimulate moments of introspection, and provide constructive feedback. Effective mentoring in STEM allows trainees, especially underrepresented minorities (URMs), to flourish in welcoming and supportive environments. However, URM trainees often experience inadequate mentoring due to their mentors inexperience with URM groups, poor mentorship training, or a lack of understanding of their mentees journey. To promote diversity, equity, and inclusion in STEM education and research, it is essential for mentors and mentees to work together with creativity, authenticity, and networking. In this workshop, we will focus on mentees perspective on how mentors can enhance their training, professional and career development, and improve their focus. We analyzed data on feedback obtained from students interested in pursuing graduate education who attended a recent workshop. Our results show that despite low initial expectations for the workshop, many students were satisfied with the knowledge they learned. The future of increasing the URM representation in STEM lies in providing adequate community support and mentorship throughout the careers of URM professionals.

scientific communication and education↗

bioRxiv2021

An effective workshop on How to be an Effective Mentor for Underrepresented STEM Trainees

Despite an increase in programming to promote persons excluded by their ethnicity or race (PEER) scholars, minorities remain underrepresented in many STEM programs. The academic pipeline is largely leaky for underrepresented minority (URM) scholars due to a lack of effective mentorship. Many URM students experience microaggressions and discrimination from their mentors due to a lack of quality mentorship training. In this workshop, we provide a framework for how to be an effective mentor to URM trainees. Mentees, especially URM trainees, can flourish in effective mentoring environments where they feel welcomed and can comfortably develop new ideas without feeling threatened by external factors. Effective mentoring environments provide motivational support, empathy, cultural competency, and training.

scientific communication and education↗

bioRxiv2021

Using a Champion-Oriented Mindset to Overcome the Challenges of Graduate School

Despite efforts to increase diversity, a glaring underrepresentation of minorities (URM) persists in the fields of science, technology, engineering, and mathematics (STEM). Graduate school can be a stressful step in the STEM pipeline, especially for students previously unaware of the structure and challenges of post-graduate education. To promote successful minority participation in STEM and prepare prospective students for the impending challenges of graduate school, we developed a workshop based on the mentoring and fostering of a champion-oriented mindset entitled, "The Trials and Tribulations of Graduate School: How Do You Make an Impact?". We administered the workshop to a cohort of university undergraduates and conducted pre- and post-workshop surveys to measure students perceived need for instruction on specific workshop topics. The results suggest that the workshop was well received by the students and provided information that they considered helpful to help navigate the graduate school process.

scientific communication and education↗

bioRxiv2021

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N=10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase (www.facebase.org, doi.org/10.25550/3-HXMC) and GitHub (https://github.com/jaydevine/MusMorph).

genetics↗

bioRxiv2021

Whole genome analysis for 163 guide RNAs in Cas9 edited mice reveals minimal off-target activity

Introductory ParagraphThe Knockout Mouse Phenotyping Program (KOMP2) uses CRISRPR/Cas9 for high-throughput mouse line production to generate null alleles in the inbred C57BL/6N strain for broad-based in vivo phenotyping. In order to assess the risk of spurious S. pyogenes Cas9-induced off-target mutagenesis, we applied whole genome sequencing to compare the genomes of 50 Cas9-derived founder mice representing 163 different gRNAs to 28 untreated inbred control mice. Our analysis pipeline detected 28 off-target sequence variants associated with 21 guides. These potential off-targets were identified in 18/50 (36%) founders with 9/28 (32%) independently validated corresponding to 8 founder animals. In total, only 4.9% (8/163) of all guides exhibited off-target activity resulting in a rate of 0.16 Cas9 off-target mutations per founder analyzed. In comparison, we observed ~1225 unique variants in each mouse regardless of whether or not it was exposed to Cas9. These findings indicate that Cas9-mediated off-target mutagenesis is rare in founder knockout mice generated using guide RNAs designed to minimize off-target risk. Overall, bona fide off-target variants comprise a small fraction of the genetic heterogeneity found in carefully maintained colonies of inbred strains.

genetics↗

bioRxiv2021

The extracellular matrix gene, Svep1, orchestrates airway patterning and the transition from lung branching morphogenesis to alveolar maturation in the mouse

Disruptions in airway branching or alveolar differentiation during lung development can lead to severe respiratory deficiencies and neonatal death. The molecular mechanisms governing branching patterning and early alveolar formation remain elusive. Loss of Svep1 function in mice results in various developmental defects, including lung hypoplasia and perinatal lethality. Our examination of the lungs of Svep1 knockout (Svep1-/-) mouse embryos, both in vivo and in vitro, revealed that Svep1 mutants exhibit an increase in the number of disorganized distal airway tips and progressively greater disruption of lung lobe morphology over time and saccular development. Svep1 interacts with FGF signaling to regulate smooth muscle differentiation and, together with Fgf9, guides airway branching patterning. Transcriptomic data from the lungs of Svep1-/- embryos revealed dysregulated gene expression affecting saccular maturation. Our findings demonstrate that Svep1 is a key extracellular matrix player shaping airway morphology and influencing alveolar fate. These insights offer potential avenues for therapeutic interventions in congenital lung disorders.

developmental biology↗

bioRxiv2020

Shapes and Genescapes: Mapping Multivariate Phenotype-Biological Process Associations for Craniofacial Shape

Realistic mappings of genes to morphology are inherently multivariate on both sides of the equation. The importance of coordinated gene effects on morphological phenotypes is clear from the intertwining of gene actions in signaling pathways, gene regulatory networks, and developmental processes underlying the development of shape and size. Yet, current approaches tend to focus on identifying and localizing the effects of individual genes and rarely leverage the information content of high dimensional phenotypes. Here, we explicitly model the joint effects of biologically coherent collections of genes on a multivariate trait--craniofacial shape -- in a sample of n = 1,145 mice from the Diversity Outbred (DO) experimental line. We use biological process gene ontology (GO) annotations to select skeletal and facial development gene sets and solve for the axis of shape variation that maximally covaries with gene set marker variation. We use our process-centered, multivariate genotype-phenotype (MGP) approach to determine the overall contributions to craniofacial variation of genes involved in relevant processes and how variation in different processes corresponds to multivariate axes of shape variation. Further, we compare the directions of effect in phenotype space of mutations to the primary axis of shape variation associated with broader pathways within which they are thought to function. Finally, we leverage the relationship between mutational and pathway-level effects to predict phenotypic effects beyond craniofacial shape in specific mutants. We also introduce an online application which provides users the means to customize their own process-centered craniofacial shape analyses in the DO. The process-centered approach is generally applicable to any continuously varying phenotype and thus has wide-reaching implications for complex-trait genetics.

genomics↗

bioRxiv2019

Human and mouse essentiality screens as a resource for disease gene discovery

Although genomic sequencing has been transformative in the study of rare genetic diseases, identifying causal variants remains a considerable challenge that can be addressed in part by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from the comprehensive viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and from human cell line essentiality screens. We propose a novel, cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing characteristics in the biological processes they regulate, tissue expression levels and human mutation rates. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented in the developmental lethal category, representing genes not essential for cell survival but required for organism development. Exploiting this finding, we have screened developmental disorder cases from three independent disease sequencing consortia and identified potentially pathogenic, de novo variants shared in different patients for several developmental lethal genes that have not previously been associated with rare disease. We therefore propose FUSIL as an efficient resource for disease gene discovery.

genomics↗

bioRxiv2018

Robust Mouse Tracking in Complex Environments using Neural Networks

The ability to track animals accurately is critical for behavioral experiments. For video-based assays, this is often accomplished by manipulating environmental conditions to increase contrast between the animal and the background, in order to achieve proper foreground/background detection (segmentation). However, as behavioral paradigms become more sophisticated with ethologically relevant environments, the approach of modifying environmental conditions offers diminishing returns, particularly for scalable experiments. Currently, there is a need for methods to monitor behaviors over long periods of time, under dynamic environmental conditions, and in animals that are genetically and behaviorally heterogeneous. To address this need, we developed a state-of-the-art neural network-based tracker for mice, using modern machine vision techniques. We test three different neural network architectures to determine their performance on genetically diverse mice under varying environmental conditions. We find that an encoder-decoder segmentation neural network achieves high accuracy and speed with minimal training data. Furthermore, we provide a labeling interface, labeled training data, tuned hyperparameters, and a pre-trained network for the mouse behavior and neuroscience communities. This general-purpose neural network tracker can be easily extended to other experimental paradigms and even to other animals, through transfer learning, thus providing a robust, generalizable solution for biobehavioral research.

animal behavior and cognition↗

bioRxiv2017

Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

Transgenesis has been a mainstay of mouse genetics for over 30 years, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported due in part to limitations in the discovery tools. Targeted Locus Amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events, and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

genetics↗