Stochastic contraction of myosin minifilaments drives evolution of microridge protrusion patterns in epithelial cells

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally elongated protrusions on mucosal epithelial cells, where they form evenly spaced, mazelike patterns that dynamically remodel by fission and fusion. To characterize how microridges form their highly ordered, subcellular patterns and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridge spacing and alignment became increasingly well ordered. Imaging F-actin and non-muscle myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked fission and fusion rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging allowed us to image individual NMII minifilaments in the apical cortex of cells in live animals, revealing that minifilaments are tethered to protrusions and often connect adjacent microridges. NMII minifilaments connecting the ends of two microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. These findings demonstrate that as cells mature, cortical NMII activity orchestrates a remodeling process that creates an increasingly orderly microridge arrangement

Tissue-Specific Transcriptomes Reveal Gene Expression Trajectories in Two Maturing Skin Epithelial Layers in Zebrafish Embryos.

Epithelial cells are the building blocks of many organs, including skin. The vertebrate skin initially consists of two epithelial layers, the outer periderm and inner basal cell layers, which have distinct properties, functions, and fates. The embryonic periderm ultimately disappears during development, whereas basal cells proliferate to form the mature, stratified epidermis. Although much is known about mechanisms of homeostasis in mature skin, relatively little is known about the two cell types in pre-stratification skin. To define the similarities and distinctions between periderm and basal skin epithelial cells, we purified them from zebrafish at early development stages and deeply profiled their gene expression. These analyses identified groups of genes whose tissue enrichment changed at each stage, defining gene flow dynamics of maturing vertebrate epithelia. At each of 52 and 72 hr post-fertilization (hpf), more than 60% of genes enriched in skin cells were similarly expressed in both layers, indicating that they were common epithelial genes, but many others were enriched in one layer or the other. Both expected and novel genes were enriched in periderm and basal cell layers. Genes encoding extracellular matrix, junctional, cytoskeletal, and signaling proteins were prominent among those distinguishing the two epithelial cell types. In situ hybridization and BAC transgenes confirmed our expression data and provided new tools to study zebrafish skin. Collectively, these data provide a resource for studying common and distinguishing features of maturing epithelia

Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples

A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites.

Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. Here, we describe an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. We found that somatosensory neurons in Drosophila and zebrafish induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons

New transgenic reporters identify somatosensory neuron subtypes in larval zebrafish

To analyze somatosensory neuron diversity in larval zebrafish, we identified several enhancers from the zebrafish and pufferfish genomes and used them to create five new reporter transgenes. Sequential deletions of three of these enhancers identified small sequence elements sufficient to drive expression in zebrafish trigeminal and Rohon-Beard (RB) neurons. One of these reporters, using the Fru.p2x3-2 enhancer, highlighted a somatosensory neuron subtype that expressed both the p2rx3a and pkcα genes. Comparison with a previously described trpA1b reporter revealed that it highlighted the same neurons as the Fru.p2x3-2 reporter. To determine whether neurons of this subtype possess characteristic peripheral branching morphologies or central axon projection patterns, we analyzed the morphology of single neurons. Surprisingly, although these analyses revealed diversity in peripheral axon branching and central axon projection, PKCα/p2rx3a/trpA1b-expressing RB cells did not possess obvious characteristic morphological features, suggesting that even within this molecularly defined subtype, individual neurons may possess distinct properties. The new transgenes created in this study will be powerful tools for further characterizing the molecular, morphological, and developmental diversity of larval somatosensory neurons

Hydrogen Peroxide Promotes Injury-Induced Peripheral Sensory Axon Regeneration in the Zebrafish Skin

Production of H2O2 by injured zebrafish skin cells promotes the regeneration of nearby somatosensory axon terminals, thus coordinating wound healing of the skin with sensory reinnervation

Developmentally Regulated Impediments to Skin Reinnervation by Injured Peripheral Sensory Axon Terminals

SummaryThe structural plasticity of neurites in the central nervous system (CNS) diminishes dramatically after initial development, but the peripheral nervous system (PNS) retains substantial plasticity into adulthood. Nevertheless, functional reinnervation by injured peripheral sensory neurons is often incomplete [1–6]. To investigate the developmental control of skin reinnervation, we imaged the regeneration of trigeminal sensory axon terminals in live zebrafish larvae following laser axotomy. When axons were injured during early stages of outgrowth, regenerating and uninjured axons grew into denervated skin and competed with one another for territory. At later stages, after the establishment of peripheral arbor territories, the ability of uninjured neighbors to sprout diminished severely, and although injured axons reinitiated growth, they were repelled by denervated skin. Regenerating axons were repelled specifically by their former territories, suggesting that local inhibitory factors persist in these regions. Antagonizing the function of several members of the Nogo receptor (NgR)/RhoA pathway improved the capacity of injured axons to grow into denervated skin. Thus, as in the CNS, impediments to reinnervation in the PNS arise after initial establishment of axon arbor structure