Gene Expression Heterogeneity in Skeletal Muscle Nuclei Ashleigh Van Deusen Molecular, Cellular and Developmental Biology University of Colorado, Boulder Defense: April 3, 2019 Thesis Advisor: Dr. Bradley B. Olwin, Department of Molecular, Cellular and Developmental Biology Defense Committee: Dr. Jennifer Martin, Honors Council, Department of Molecular, Cellular, and Developmental Biology Dr. Bradley B. Olwin Department of Molecular, Cellular and Developmental Biology Dr. Alison Vigers, Department of Molecular, Cellular and Developmental Biology and Neuroscience Dr. Heidi Day, Department of Neuroscience 2 Table of Contents Abstract Introduction Chapter 1: Single Muscle Nuclear Isolation and Characterization I. Introduction II. Methods III. Results IV. Discussion Chapter 2: Single Nucleus RNA Sequencing in Skeletal Muscle I. Introduction II. Methods III. Results IV. Discussion General Discussion 3 Abstract Skeletal muscle is required for breathing and voluntary movement and loss of muscle mass is a significant contributor to humans’ inability to stay active throughout their entire lifespan. Skeletal muscle consists of multinucleated cells and there is no comprehensive data on the gene expression of the hundreds of nuclei located in a single myofiber required for contraction, attachment to tendons and force generation. Typical methodologies to examine gene expression of single cells cannot be performed on skeletal muscle because individual cells are large and multinucleated. Single nucleus sequencing can be exploited to examine differential gene expression that likely occurs among the hundreds of myonuclei organized within the myofiber. Isolation of nuclei from skeletal muscle previously done masked variability that exists in different muscles and types. I devised a protocol to isolate nuclei from a single muscle. I validated the method and performed it in multiple muscle groups, allowing the study of different expression profiles from a single mouse. The established protocol was employed in conjunction with single nucleus RNA Sequencing to investigate differential gene expression in the nuclei of adult, aged, and injured muscle. Preliminary analysis of adult muscle indicates clusters of myonuclei and mononucleated cells. There was widespread misregulation in aged muscle, myonuclei genes were expressed in all clusters and some of the mononucleated populations were undetected. Satellite cell nuclei present in uninjured aged muscle have become activated, impairing the longevity of muscle maintenance. This work could provide new therapeutic targets to prevent the age induced loss of muscle regeneration. 4 Introduction Aging is an inevitable part of being human. The goal of a large body of research is attempting to better understand aging so ultimately it can be delayed. For most, a significant portion of the 79-year average lifetime will not be lived actively or disease free. The average health span is nearly 20% shorter than overall lifespan at about 63.3 years, meaning individuals are unable to perform desired activities (World Health Organization, 2016). Interventions that slow the types of processes that limit health span would be a major step towards helping people live longer, healthier lives. One approach to delay aging targets muscle wasting, a major contributor that shortens health span. Sarcopenia is the age-associated loss of function and muscle mass unrelated to disease, but leads to loss of strength, immobility and frailty. Both men and women experience sarcopenia, which results in 20-40% loss in strength for adults in their seventies and eighties (Doherty, 2003). All organ systems are affected by aging and understanding the distinct changes that occur in skeletal muscle as humans age could lead to new therapeutic targets to combat the impacts of aging. Nearly 40% of human body mass is skeletal muscle, which is required for voluntary movement. Skeletal muscle consists of two distinct muscle cell populations, satellite cells, and the terminally differentiated multinucleated myofibers (Dumont et al., 2015; Zeng et al., 2016). Proper functioning of these cells is dependent on gene expression regulation, but until recently cell to cell variation has been difficult to study. Recent advances in high-throughput sequencing technologies makes studying gene expression regulation more accessible. In particular, RNA sequencing (RNA-Seq) has allowed the investigation of differential gene expression in tissues. The method is advantageous for studying RNA because it is more sensitive to subtle differences by capturing both known and novel features and having a specific target is not required 5 (Bengston et al., 2018). A comprehensive view of gene expression is provided, all of the transcripts are averaged from the entire sample so cell to cell variation is lost. Single cell RNA- seq (scRNA-Seq) and single nucleus RNA-seq (snRNA-Seq) have unique advantages over RNA- seq by allowing examination of cell to cell variation and eliminating the possible masked heterogeneity of bulk samples (Illumina, 2018). Heterogeneity in gene expression is the variation of expression between cells and is masked in bulk samples because averages are taken from all of the cells, so individual cell-to-cell variation is not exposed. Single cell and single nucleus sequencing provide information into any variation that occurs in a sample and allow the study of complex tissues in environmental or time-dependent processes. Examining what is actively being transcribed in the nucleus, versus transcripts in the nucleus and already existing transcripts in the cytoplasm, can help identify changes that may occur during aging. Single nucleus sequencing could be useful in identifying factors that contribute to muscle wasting, but isolation needs to be performed in a single muscle. Skeletal muscle fibers exist in a multinucleated syncytium surrounded by tough connective tissue making isolation of nuclei difficult and yields low(Kuehl, 1975). Previous isolation protocols in skeletal muscle have only used pooled samples, either from multiple muscle groups in a single mouse or multiple mice but this limits the variability that would be exposed in a single muscle. I sought to devise a protocol to isolate nuclei from a single muscle. The method was validated and performed in multiple muscle groups, allowing the possibility to study many different states and expression profiles from a single mouse. The established protocol was employed in conjunction with snRNA-Seq to investigate the differential gene expression that occurs during age and injury in the hindlimb muscles of individual mice. The goal of my study was to examine the heterogeneity of nuclei in a single 6 muscle, predicting that the sequencing would uncover a few nuclei that may be large contributors to the aging phenotype. The nuclei are clustered into populations based on their gene expression and differences in the aged muscle were determined by examining the populations in the adult muscle. I expected to see many nuclear populations in the muscle including myogenic cells, satellite cells, neuromuscular junction (NMJ), myotendinous junction, and non-muscle cell types endothelial, immune, fibroblasts. I also expected mis-regulations in nuclei of the aged muscle present in one or all of the given populations. The atlas that was created of the adult, aged and injured muscles allowed me to determine the extent of heterogeneity in muscle and non-muscle nuclei and whether aged muscle differs from young adult muscle. 7 Chapter 1: Single Muscle Nuclear Isolation and Characterization Introduction Skeletal muscle is required for breathing and voluntary movement. Investigation of gene expression in a skeletal muscle lends insight into cellular needs and how they can be mis- regulated. Previous analysis methods have not been able to identify distinct cell populations coming from a single mouse or muscle, they have been in bulk providing only averages. There is need for a unique tool that could show nuclear specific transcripts and what is being actively transcribed for the cell. A nuclear isolation method from a single muscle would provide the ability to detect mis-regulations in gene expression that occur in aging and provide targets to combat dysfunction. The isolation of nuclei is advantageous for studying active transcription, and has been performed in the brain (Matevossian and Akbarian, 2008). Nuclei have been isolated in bulk skeletal muscle samples (Cutler et al., 2017) but has not been performed on a single muscle. Nuclear isolation from skeletal muscle is particularly difficult because there is a lower concentration of nuclei than in other cell types, all of the nuclei share a cytoplasm in each myofiber, and individual myofibers are held together with connective tissue making the muscle tough to break apart and release nuclei without damaging them (Fig. 1) (Kuehl, 1975). An investigation of the different nuclear populations in a single skeletal muscle could inform us about heterogenous transcription among myonuclei and what occurs in diseased and aged states. 8 Skeletal muscle consists of two distinct muscle cell populations, satellite cells, and the terminally differentiated multinucleated myofibers. Myofibers contain the contractile unit of muscle and form an ordered structure with connective tissue (Fig. 1A). Myofibers are created by hundreds of mononucleated myocytes, differentiated myoblasts created from satellite cells, that fuse together and exist in a syncytium. Once in a syncytium the myofibers are