Biology subjects

Johnson, A. N.

Publications and source records attributed to Johnson, A. N..

12 recordsLinked to original sources

bioRxiv2026

Transient activation of potent progenitor cells is required for spinal cord regeneration

Adult zebrafish exhibit full recovery following spinal cord injury. Transient expansion of stem cell-like progenitors is thought to underlie their regenerative capacity. Yet, our understanding of the identities and contributions of the crucial stem cell populations that direct spontaneous neural repair remains limited. Moreover, while most neural regeneration research is centered on promoting proliferative repair, the regulatory mechanisms that reinstate quiescence post-repair are unknown. Here, we determined the molecular identities and cellular contributions of sox2+ progenitors during spinal cord repair. Genetic lineage tracing shows zebrafish spinal progenitors, while quiescent in uninjured tissues, self-renew and differentiate into neurons and glia after injury. By single-cell sequencing, sox2+ cells are heterogeneous and biased towards neuronal or glial fates in both homeostatic and regenerating tissues. By screening for transcription factors that are differentially expressed in acute versus chronic spinal cord injury, we find the Bach1 transcription factors control transient progenitor cell activation by acting as dual activators and repressors of sox2 expression. This study elucidates the molecular diversity and contributions of sox2 expressing cells during spinal cord repair and identifies a transcriptional regulatory switch by which progenitor cells expand after injury and restore quiescence after regeneration is completed.

neuroscience↗

bioRxiv2025

Hb-EGF directs systemic muscle repair

Regenerative capacity varies between tissues, species, and stages of the life cycle. What is less appreciated is that regenerative capacity also varies with the magnitude of the injury, even within a single tissue. Vertebrate skeletal muscle efficiently regenerates following minor injuries; however, extensive injuries may result in incomplete repair, which can be debilitating. To understand if small- and large-scale muscle injuries activate distinct regenerative programs, we developed a systemic muscle injury model in zebrafish. Transcriptomic analysis of muscle and non-muscle tissues revealed that systemic and local muscle injuries elicit distinct molecular responses, both quantitatively and qualitatively. Systemic muscle injury activated the expression of Heparin binding epidermal-like growth factor (Hb-EGF) in the epidermis, and Hb-EGF is necessary for systemic muscle repair. Conversely, local muscle injury did not induce Hb-EGF expression and Hb-EGF was not required for local muscle repair. These studies suggest that large- and small-scale muscle injuries activate different regenerative programs, resulting in either systemic or local repair.

developmental biology↗

bioRxiv2025

Expanding Targeted Instrumentation for Discovery Applications: Complement Reporter Ion Quantification with a Quadrupole-Ion Trap Instrument

Proteomics workflows have traditionally been divided into discovery-based and targeted approaches, with instrumentation optimized specifically for each. Discovery experiments typically utilize high-resolution analyzers while targeted workflows rely on the sensitivity and specificity of triple quadrupole systems. Recently, a quadrupole-ion trap instrument (Stellar MS) demonstrated superior performance for targeted applications compared to conventional triple quadrupoles. In this study, we expand the capabilities of this platform to multiplexed shotgun proteomics using complement reporter ion quantification in an ion trap (iTMTproC). Benchmarking experiments with defined standards show that iTMTproC achieves quantification accuracy and interference reduction comparable to MultiNotch MS3 on the Orbitrap Fusion Lumos, a dedicated quadrupole-ion trap-Orbitrap tribrid instrument optimized for this purpose. Notably, iTMTproC quantifies slightly more proteins than MultiNotch MS3. We further validate this approach through a developmental time-series analysis of frog embryos, obtaining proteomic data nearly indistinguishable from MultiNotch MS3, with slightly increased protein quantification depth. These findings significantly extend the functionality of targeted instrumentation, underscoring the versatility of quadrupole-ion trap systems and providing cost-effective access to highly accurate, multiplexed quantitative shotgun proteomics.

systems biology↗

bioRxiv2024

Elevated phagocytic capacity directs innate spinal cord repair

Immune cells elicit a continuum of transcriptional and functional states after spinal cord injury (SCI). In mammals, inefficient debris clearance and chronic inflammation impede recovery and overshadow pro-regenerative immune functions. We found that, unlike mammals, zebrafish SCI elicits transient immune activation and efficient debris clearance, without causing chronic inflammation. Single-cell transcriptomics and inducible genetic ablation showed zebrafish macrophages are highly phagocytic and required for regeneration. Cross-species comparisons between zebrafish and mammalian macrophages identified transcription and immune response regulator (tcim) as a macrophage-enriched zebrafish gene. Genetic deletion of zebrafish tcim impairs phagocytosis and regeneration, causes aberrant and chronic immune activation, and can be rescued by transplanting wild-type immune precursors into tcim mutants. Conversely, genetic expression of human TCIM accelerates debris clearance and regeneration by reprogramming myeloid precursors into activated phagocytes. This study establishes a central requirement for elevated phagocytic capacity to achieve innate spinal cord repair.

neuroscience↗

bioRxiv2024

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development, and promoting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility from the C-zone? Globular domains critical to MyBP-Cs modulatory functions are absent from MyBP-H, suggesting MyBP-H may be functionally silent. However, our results suggest an active role. Small angle x-ray diffraction of intact larval tails revealed MyBP-H contributes to the compression of the myofilament lattice accompanying stretch or contraction, while in vitro motility experiments indicate MyBP-H shares MyBP-Cs capacity as a molecular "brake". These results provide new insights and raise questions about the role of the C-zone during muscle development.

biophysics↗

bioRxiv2023

An in vivo platform to identify clinically relevant pathogenic loci using Drosophila melanogaster

Rare genetic disease discovery efforts typically lead to the identification of new disease genes. PreMIER (Precision Medicine Integrated Experimental Resources) is a collaborative platform designed to facilitate functional evaluation of human genetic variants in model systems, and to date the PreMIER Consortium has evaluated over 50 variants in patients with genetic disorders. To understand if Drosophila could be used to identify pathogenic disease loci as part of the PreMIER Consortium, we used tissue-specific gene knockdown in the fly as a proof of principle experiment. Tissue-specific knockdown of seven conserved disease genes caused significant changes in viability, longevity, behavior, motor function, and neuronal survival arguing a set of defined assays can be used to determine if a gene of uncertain significance (GUS) regulates specific physiological processes. This study highlights the utility of a tissue-specific knockdown platform in Drosophila to characterize GUS, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders

physiology↗

bioRxiv2022

The serine/threonine kinase Back seat driver prevents cell fusion to maintain cell identity

Cell fate specification is essential for every major event of embryogenesis, and subsequent cell maturation ensures individual cell types acquire specialized functions. The mechanisms that regulate cell fate specification have been studied exhaustively, and each technological advance in developmental biology ushers in a new era of studies aimed at uncovering the most fundamental processes by which cells acquire unique identities. What is less appreciated is that mechanisms are in place to ensure cell identity is maintained throughout the life of the organism. The body wall musculature in the Drosophila embryo is a well-established model to study cell fate specification, as each hemisegment in the embryo generates and maintains thirty muscles with distinct identities. Here we show the serine/threonine kinase Back seat driver (Bsd), which regulates muscle morphogenesis, also maintains cell identity. Once specified, the thirty body wall muscles fuse with mononucleate muscle precursors that lack a specific identity to form multinucleate striated muscles. Importantly, body wall muscles do not fuse with each other and thereby maintain distinct identities. We show that Bsd prevents inappropriate fusion among the thirty body wall muscles. Thus, the regulation of cell fusion is one mechanism that maintains cell identity.

developmental biology↗

bioRxiv2021

An in vivo approach to characterize novel variants associated with musculoskeletal disorders

Nemaline Myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as Cap Myopathy, congenital fiber type disproportion, and distal arthrogrypsosis (DA). TPM2-related disorders have not been modeled in vivo, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found two variants, K49Del and E122K, significantly affected muscle morphogenesis and muscle function, in part by disrupting myotube guidance. Transient overexpression of K49Del and E122K also disrupted the morphogenesis of immortalized mouse myoblasts in vitro, and negatively affected zebrafish muscle development and function in vivo. We used our transient overexpression assays in zebrafish to characterize two novel TPM2 variants that we identified in DA patients (V129A and E139K), and found these variants caused musculoskeletal defects similar to those of the known pathogenic variants. In addition, the severity of musculoskeletal phenotypes in zebrafish expressing TPM2 variants correlated with the severity of clinical phenotypes observed in DA patients. Our study establishes transient overexpression in zebrafish as an efficient platform to characterize variants of uncertain significance in TPM2 in vivo, and suggests that this method can be used to predict the clinical severity of variants associated with DA and congenital myopathies.

genetics↗

bioRxiv2021

A genetic screen for regulators of muscle morphogenesis in Drosophila

The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendons. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including bsd, kon, ths, and tum, arguing our screening strategy was effective and efficient. We also identified and sequenced new alleles of salm, barr, and ptc that presumably disrupt independent pathways directing muscle morphogenesis. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be identified. This screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that determine skeletal muscle topology.

developmental biology↗

bioRxiv2020

SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome

Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/423533v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1be5b38org.highwire.dtl.DTLVardef@56e061org.highwire.dtl.DTLVardef@1a52040org.highwire.dtl.DTLVardef@1e86751_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryInfections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show a number of non-neural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood, so we developed three models to investigate the impact of neuroinflammation on muscle performance. We found that bacterial infection, COVID-like viral infection, and expression of a neurotoxic protein associated with Alzheimer' s disease promoted the accumulation of reactive oxygen species (ROS) in the brain. Excessive ROS induces the expression of the cytokine Unpaired 3 (Upd3) in insects, or its orthologue IL-6 in mammals, and CNS-derived Upd3/IL-6 activates the JAK/Stat pathway in skeletal muscle. In response to JAK/Stat signaling, mitochondrial function is impaired and muscle performance is reduced. Our work uncovers a brain-muscle signaling axis in which infections and chronic diseases induce cytokine-dependent changes in muscle performance, suggesting IL-6 could be a therapeutic target to treat muscle weakness caused by neuroinflammation.

microbiology↗

bioRxiv2020

Post-mitotic myotubes repurpose the cytokinesis machinery to effect cellular guidance

Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. Here we show the mitotic kinase Polo, which regulates all steps of mitosis [1-3], also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis [4-6]. We show that in the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases.

cell biology↗

bioRxiv2018

Intrinsic cooperativity potentiates parallel cis-regulatory evolution

Convergent evolutionary events in independent lineages provide an opportunity to understand why evolution favors certain outcomes over others. We studied such a case, where a large set of genes--those coding for the ribosomal proteins--gained cis-regulatory sequences for a particular transcription regulator (Mcm1) in independent fungal lineages. We present evidence that these gains occurred because Mcm1 shares a mechanism of transcriptional activation with an ancestral regulator of the ribosomal protein genes, Rap1. Specifically, we show that Mcm1 and Rap1 have the inherent ability to cooperatively activate transcription through contacts with the general transcription factor TFIID. Because the two regulatory proteins share a common interaction partner, the presence of one ancestral cis-regulatory sequence can \"channel\" random mutations into functional sites for the second regulator. At a genomic scale, this type of intrinsic cooperativity can account for a pattern of parallel evolution involving the fixation of hundreds of substitutions.

evolutionary biology↗