Transcriptional regulation and alternative splicing cooperate in muscle fiber-type specification in flies and mammals

Muscles coordinate body movements throughout the animal kingdom. Each skeletal muscle is built of large, multi-nucleated cells, called myofibers, which are classified into several functionally distinct types. The typical fiber-type composition of each muscle arises during development, and in mammals is extensively adjusted in response to postnatal exercise. Understanding how functionally distinct muscle fiber-types arise is important for unraveling the molecular basis of diseases from cardiomyopathies to muscular dystrophies. In this review, we focus on recent advances in Drosophila and mammals in understanding how muscle fiber-type specification is controlled by the regulation of transcription and alternative splicing. We illustrate the cooperation of general myogenic transcription factors with muscle fiber-type specific transcriptional regulators as a basic principle for fiber-type specification, which is conserved from flies to mammals. We also examine how regulated alternative splicing of sarcomeric proteins in both flies and mammals can directly instruct the physiological and biophysical differences between fiber-types. Thus, research in Drosophila can provide important mechanistic insight into muscle fiber specification, which is relevant to homologous processes in mammals and to the pathology of muscle diseases. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved

A Versatile Two-Step CRISPR- and RMCE-Based Strategy for Efficient Genome Engineering in Drosophila

The development of clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technologies promises a quantum leap in genome engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila melanogaster are still restricted to a few genes, use variable experimental conditions, and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different locations in the genome, including a gene on the fourth chromosome, at comparably high efficiencies. Our data suggest that any fly laboratory can engineer their favorite gene for a broad range of applications within approximately 3 months

Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo

Muscle forces are produced by repeated stereotypical actomyosin units called sarcomeres. Sarcomeres are chained into linear myofibrils spanning the entire muscle fiber. In mammalian body muscles, myofibrils are aligned laterally, resulting in their typical cross-striated morphology. Despite this detailed textbook knowledge about the adult muscle structure, it is still unclear how cross-striated myofibrils are built in vivo. Here, we investigate the morphogenesis of Drosophila abdominal muscles and establish them as an in vivo model for cross-striated muscle development. By performing live imaging, we find that long immature myofibrils lacking a periodic actomyosin pattern are built simultaneously in the entire muscle fiber and then align laterally to give mature cross-striated myofibrils. Interestingly, laser micro-lesion experiments demonstrate that mechanical tension precedes the formation of the immature myofibrils. Moreover, these immature myofibrils do generate spontaneous Ca2+-dependent contractions in vivo, which, when chemically blocked, result in cross-striation defects. Taken together, these results suggest a myofibrillogenesis model in which mechanical tension and spontaneous muscle twitching synchronize the simultaneous self-organization of different sarcomeric protein complexes to build highly regular cross-striated myofibrils spanning the length of large muscle fibers

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Synopsis image Glial cell line derived neurotrophic factor (GDNF) improves survival in toxin-models of Parkinson's disease and is currently undergoing clinical development, however the protective mechanism is elusive. This study provides evidence that the GDNF receptor Ret rescues defects of a genetic Parkinson model and proposes a new mechanism-of-action. Active Ret overexpression rescues muscle degeneration and mitochondrial morphology in muscles and dopamine neurons in Pink1 mutant Drosophila. In human neuroblastoma cells, GDNF treatment rescues mitochondrial fragmentation caused by Pink1 knockdown. Ret signaling improves mitochondrial respiration and activity of complex I, providing a potential novel mechanism for the protective effect of GDNF/Ret. Abstract Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (Ret(MEN)(2B)) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret(MEN)(2B) significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD

The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle.

In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis

A genome-wide resource for the analysis of protein localisation in Drosophila

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts

Vestigial Is Required during Late-Stage Muscle Differentiation in Drosophila melanogaster Embryos

The Drosophila member of the vestigial-like gene family (vestigial) is known primarily as a transcriptional activator that defines cell identity during Drosophila wing differentiation. We show that during embryo development Vestigial also has a role during specification of muscle–muscle attachments in ventral longitudinal muscles

An IFN-γ-IL-18 Signaling Loop Accelerates Memory CD8+ T Cell Proliferation

Rapid proliferation is one of the important features of memory CD8+ T cells, ensuring rapid clearance of reinfection. Although several cytokines such as IL-15 and IL-7 regulate relatively slow homeostatic proliferation of memory T cells during the maintenance phase, it is unknown how memory T cells can proliferate more quickly than naïve T cells upon antigen stimulation. To examine antigen-specific CD8+ T cell proliferation in recall responses in vivo, we targeted a model antigen, ovalbumin(OVA), to DEC-205+ dendritic cells (DCs) with a CD40 maturation stimulus. This led to the induction of functional memory CD8+ T cells, which showed rapid proliferation and multiple cytokine production (IFN-γ, IL-2, TNF-α) during the secondary challenge to DC-targeted antigen. Upon antigen-presentation, IL-18, an IFN-γ-inducing factor, accumulated at the DC:T cell synapse. Surprisingly, IFN-γ receptors were required to augment IL-18 production from DCs. Mice genetically deficient for IL-18 or IFN-γ-receptor 1 also showed delayed expansion of memory CD8+ T cells in vivo. These results indicate that a positive regulatory loop involving IFN-γ and IL-18 signaling contributes to the accelerated memory CD8+ T cell proliferation during a recall response to antigen presented by DCs

A developmental approach to predicting neuronal connectivity from small biological datasets: a gradient-based neuron growth model.

PMCID: PMC3931784 Open Access article: BB/G006652/1 and BB/G006369/1.Relating structure and function of neuronal circuits is a challenging problem. It requires demonstrating how dynamical patterns of spiking activity lead to functions like cognitive behaviour and identifying the neurons and connections that lead to appropriate activity of a circuit. We apply a "developmental approach" to define the connectome of a simple nervous system, where connections between neurons are not prescribed but appear as a result of neuron growth. A gradient based mathematical model of two-dimensional axon growth from rows of undifferentiated neurons is derived for the different types of neurons in the brainstem and spinal cord of young tadpoles of the frog Xenopus. Model parameters define a two-dimensional CNS growth environment with three gradient cues and the specific responsiveness of the axons of each neuron type to these cues. The model is described by a nonlinear system of three difference equations; it includes a random variable, and takes specific neuron characteristics into account. Anatomical measurements are first used to position cell bodies in rows and define axon origins. Then a generalization procedure allows information on the axons of individual neurons from small anatomical datasets to be used to generate larger artificial datasets. To specify parameters in the axon growth model we use a stochastic optimization procedure, derive a cost function and find the optimal parameters for each type of neuron. Our biologically realistic model of axon growth starts from axon outgrowth from the cell body and generates multiple axons for each different neuron type with statistical properties matching those of real axons. We illustrate how the axon growth model works for neurons with axons which grow to the same and the opposite side of the CNS. We then show how, by adding a simple specification for dendrite morphology, our model "developmental approach" allows us to generate biologically-realistic connectomes

Multiple Dendritic Cell Populations Activate CD4+ T Cells after Viral Stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8α DC play a prominent, and sometimes exclusive, role in driving amplification of CD8+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4+ and CD8+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8α DC populations in the amplification of CD8+ and CD4+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8+ T cells are dominated by presentation of antigen by CD8α DC but can involve non-CD8α DC. In contrast, CD4+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity