Neuronal identity has long been thought of as immutable, so that once a cell acquires a specific fate, it is maintained for life.(1) Studies using the overexpression of potent transcription factors to experimentally reprogram neuronal fate in the mouse neocortex(2,3) and retina(4,5) have challenged this notion by revealing that post-mitotic neurons can switch their identity. Whether fate reprogramming is part of normal development in the central nervous system (CNS) is unclear. While there are some reports of physiological cell fate reprogramming in invertebrates,(6,7) and in the vertebrate peripheral nervous system,(8) endogenous fate reprogramming in the vertebrate CNS has not been documented. Here, we demonstrate spontaneous fate re-specification in an interneuron lineage in the zebrafish retina. We show that the visual system homeobox 1 (vsx1)-expressing lineage, which has been associated exclusively with excitatory bipolar cell (BC) interneurons,(9-12) also generates inhibitory amacrine cells (ACs). We identify a role for Notch signaling in conferring plasticity to nascent vsx1 BCs, allowing suitable transcription factor programs to re-specify them to an AC fate. Overstimulating Notch signaling enhances this physiological phenotype so that both daughters of a vsx1 progenitor differentiate into ACs and partially differentiated vsx1 BCs can be converted into ACs. Furthermore, this physiological re-specification can be mimicked to allow experimental induction of an entirely distinct fate, that of retinal projection neurons, from the vsx1 lineage. Our observations reveal unanticipated plasticity of cell fate during retinal development.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Conventionally, neuronal development is regarded to follow a stereotypic sequence of neurogenesis, migration, and differentiation. We demonstrate that this notion is not a general principle of neuronal development by documenting the timing of mitosis in relation to multiple differentiation events for bipolar cells (BCs) in the zebrafish retina using in vivo imaging. We found that BC progenitors undergo terminal neurogenic divisions while in markedly disparate stages of neuronal differentiation. Remarkably, the differentiation state of individual BC progenitors at mitosis is not arbitrary but matches the differentiation state of post‐mitotic BCs in their surround. By experimentally shifting the relative timing of progenitor division and differentiation, we provide evidence that neurogenesis and differentiation can occur independently of each other. We propose that the uncoupling of neurogenesis and differentiation could provide neurogenic programs with flexibility, while allowing for synchronous neuronal development within a continuously expanding cell pool.

Improved expansion microscopy provides a super-resolution method that is accessible to any laboratory with a fluorescence microscope.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Objectives: To characterize phenotypes of T cells that accumulated in multiple sclerosis (MS) lesions, to compare the lesional T-cell receptor (TCR) repertoire of T-cell subsets to peripheral blood, and to identify paired a and beta chains from single CD8(+) T cells from an index patient who we followed for 18 years.Methods: We combined immunohistochemistry, laser microdissection, and single-cell multiplex PCR to characterize T-cell subtypes and identify paired TCR alpha and TCR beta chains from individual brain-infiltrating T cells in frozen brain sections. The lesional and peripheral TCR repertoires were analyzed by pyrosequencing.Results: We found that a TCR V beta 1' T-cell population that was strikingly expanded in active brain lesions at clinical onset comprises several subclones expressing distinct yet closely related V alpha 7.2(+) alpha chains, including a canonical V alpha 7.2-J alpha 33 chain of mucosal-associated invariant T (MAIT) cells. Three other a chains bear striking similarities in their antigen-recognizing, hypervariable complementarity determining region 3. Longitudinal repertoire studies revealed that the TCR chains that were massively expanded in brain at onset persisted for several years in blood or CSF but subsequently disappeared except for the canonical V alpha 7.2(+) MAIT cell and a few other TCR sequences that were still detectable in blood after 18 years.Conclusions: Our observation that a massively expanded TCR V beta 1-J beta 2.3 chain paired with distinct yet closely related canonical or atypical MAIT cell-related a chains strongly points to an antigen-driven process in early active MS brain lesions.

ZebrafishVol. 11, No. 6 TechnoFishCentrinFish Permit the Visualization of Centrosome Dynamics in a Cellular Context In VivoPeter Engerer, Takeshi Yoshimatsu, Sachihiro C. Suzuki, and Leanne GodinhoPeter EngererInstitute of Neuronal Cell Biology, Technische Universität München, Munich, Germany.Search for more papers by this author, Takeshi YoshimatsuDepartment of Biological Structure, University of Washington, Seattle, Washington.Search for more papers by this author, Sachihiro C. SuzukiDepartment of Biological Structure, University of Washington, Seattle, Washington.Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan.Search for more papers by this author, and Leanne GodinhoInstitute of Neuronal Cell Biology, Technische Universität München, Munich, Germany.Search for more papers by this authorPublished Online:3 Dec 2014https://doi.org/10.1089/zeb.2014.1503AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"CentrinFish Permit the Visualization of Centrosome Dynamics in a Cellular Context In Vivo." Zebrafish, 11(6), pp. 586–587FiguresReferencesRelatedDetails Volume 11Issue 6Dec 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Peter Engerer, Takeshi Yoshimatsu, Sachihiro C. Suzuki, and Leanne Godinho.CentrinFish Permit the Visualization of Centrosome Dynamics in a Cellular Context In Vivo.Zebrafish.Dec 2014.586-587.http://doi.org/10.1089/zeb.2014.1503Published in Volume: 11 Issue 6: December 3, 2014Online Ahead of Print:November 11, 2014PDF download

Adaptive optics microscopy using a de-scanned, laser-induced guide star and direct wavefront sensing allows high numerical aperture diffraction-limited imaging of fine dynamic structures deep in the intact living zebrafish brain. Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

Autoantibody-induced cellular signaling mechanisms contribute to the pathogenesis of autoimmune blistering skin disease pemphigus vulgaris (PV). Recently, it was proposed that epidermal growth factor receptor (EGFR) might be involved in PV signaling pathways. In this study, we investigated the role of EGFR by comparing the effects of epidermal growth factor (EGF) and PV-IgG on the immortalized human keratinocyte cell line HaCaT, and primary normal human keratinocytes. In contrast to EGF treatment, PV-IgG neither caused the canonical activation of EGFR via phosphorylation at tyrosine (Y)1173 followed by internalization of EGFR nor the phosphorylation of the EGFR at the c-Src-dependent site Y845. Nevertheless, both PV-IgG and EGF led to cell dissociation and cytokeratin retraction in keratinocyte monolayers. Moreover, the effects of EGF were blocked by inhibition of EGFR and c-Src whereas the effects of PV-IgG were independent of both signaling pathways. Similarly, laser tweezer experiments revealed that impaired bead binding of epidermal cadherins desmoglein (Dsg) 3 and Dsg 1 in response to PV-IgG was not affected by inhibition of either EGFR or c-Src. In contrast, EGF treatment did not interfere with Dsg bead binding. Taken together, our study indicates that the loss of Dsg-mediated adhesion and keratinocyte dissociation in pemphigus is independent of EGFR. Moreover, the mechanisms by which both EGF and PV-IgG lead to keratinocyte dissociation and cytokeratin retraction appear to be different.

The release of transmitter-filled vesicles from presynaptic terminals is a key step of neurotransmission. Prior to release, synaptic vesicles get clustered at a specialized patch of the presynaptic membrane, here referred to as the active zone. So far, mainly biochemical regulations at the active zone were regarded as decisive for synaptic vesicle clustering and release. However, using biophysical approaches, a recent paper [Siechen, et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, 12611-12616] indicated also that the micromechanical regulations within axon and terminal could be crucial for proper vesicle clustering. The authors demonstrated that the synaptic vesicle accumulations vanished after axotomy but were restored after the application of physical tension. Furthermore, axons seem to be under an intrinsic tension, which could be perceived and tuned by an axon-internal tension sensing mechanism. Therefore, mechanical force could steer vesicle clustering and consequently synapse function. Here, we review this interdisciplinary study of Siechen, et al. [Proc. Natl. Acad. Sci. U.S.A. 106, 12611-12616 (2009)] and discuss the significance of cellular mechanics on synaptic function.