The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia-like cells, actively proliferating cells, and label-retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow-dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100beta), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA-NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow-cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA-positive cells in fast domains further display a morphology distinct from radial glia and co-express PSA-NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA-NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ.
Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an ‘eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions.
Formation of organ-specific vasculatures requires cross-talk between developing tissue and specialized endothelial cells. Here we show how developing zebrafish spinal cord neurons coordinate vessel growth through balancing of neuron-derived Vegfaa, with neuronal sFlt1 restricting Vegfaa-Kdrl mediated angiogenesis at the neurovascular interface. Neuron-specific loss of flt1 or increased neuronal vegfaa expression promotes angiogenesis and peri-neural tube vascular network formation. Combining loss of neuronal flt1 with gain of vegfaa promotes sprout invasion into the neural tube. On loss of neuronal flt1, ectopic sprouts emanate from veins involving special angiogenic cell behaviours including nuclear positioning and a molecular signature distinct from primary arterial or secondary venous sprouting. Manipulation of arteriovenous identity or Notch signalling established that ectopic sprouting in flt1 mutants requires venous endothelium. Conceptually, our data suggest that spinal cord vascularization proceeds from veins involving two-tiered regulation of neuronal sFlt1 and Vegfaa via a novel sprouting mode.
Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a “black box”. Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO2), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo. To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.
Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a-and gata3-dependent precursors coexpressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses.
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