Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. ...Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.
Development of the zebrafish hypothalamus Machluf, Yossy; Gutnick, Amos; Levkowitz, Gil
Annals of the New York Academy of Sciences,
March 2011, Letnik:
1220, Številka:
1
Journal Article
Recenzirano
Hypothalamic neurons regulate fundamental body functions including sleep, blood pressure, temperature, hunger and metabolism, thirst and satiety, stress, and social behavior. This is achieved by ...means of the secretion of various hypothalamic neuropeptides and neurotransmitters that affect endocrine, metabolic, and behavioral activities. Developmental impairments of hypothalamic neuronal circuits are associated with neurological disorders that disrupt both physiological and psychological homeostasis. Hypothalamic cell specification and morphogenesis can be uniquely studied in zebrafish, a vertebrate organism readily amenable to genetic manipulations. As embryos are optically transparent and develop externally, they provide a powerful tool for in vivo analyses of neurons and their circuits. Here, we discuss the current knowledge regarding the neuroanatomy of the zebrafish hypothalamus and recent studies identifying critical determinants of hypothalamic differentiation. Taken together, these reports demonstrate that the molecular pathways underlying development of the hypothalamus are largely conserved between zebrafish and mammals. We conclude that the zebrafish has proved itself a valuable vertebrate model for understanding the patterning, specification, morphogenesis, and subsequent function of the hypothalamus.
Microtubule-based transport provides mitochondria to distant regions of neurons and is essential for neuronal health. To identify compounds that increase mitochondrial motility, we screened 1,641 ...small-molecules in a high-throughput screening platform. Indirubin and cantharidin increased mitochondrial motility in rat cortical neurons. Cantharidin is known to inhibit protein phosphatase 2A (PP2A). We therefore tested two other inhibitors of PP2A: LB-100 and okadaic acid. LB-100 increased mitochondrial motility, but okadaic acid did not. To resolve this discrepancy, we knocked down expression of the catalytic subunit of PP2A (PP2CA). This long-term inhibition of PP2A more than doubled retrograde transport of axonal mitochondria, confirming the importance of PP2A as a regulator of mitochondrial motility and as the likely mediator of cantharidin's effect.
The hypothalamo-neurohypophyseal system (HNS) is the neurovascular structure through which the hypothalamic neuropeptides oxytocin and arginine-vasopressin exit the brain into the bloodstream, where ...they go on to affect peripheral physiology. Here, we investigate the molecular cues that regulate the neurovascular contact between hypothalamic axons and neurohypophyseal capillaries of the zebrafish. We developed a transgenic system in which both hypothalamic axons and neurohypophyseal vasculature can be analyzed in vivo. We identified the cellular organization of the zebrafish HNS as well as the dynamic processes that contribute to formation of the HNS neurovascular interface. We show that formation of this interface is regulated during development by local release of oxytocin, which affects endothelial morphogenesis. This cell communication process is essential for the establishment of a tight axovasal interface between the neurons and blood vessels of the HNS. We present a unique example of axons affecting endothelial morphogenesis through secretion of a neuropeptide.
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► Live imaging of neurovascular interfaces reveals mechanisms driving morphogenesis ► Hypothalamic neurons are necessary for pituitary vascular organization ► Oxytocin signaling regulates the neurovascular interface in the pituitary
We have explored the effects of robust neural plate patterning signals, such as canonical Wnt, on the differentiation and configuration of neuronal subtypes in the zebrafish diencephalon at ...single-cell resolution. Surprisingly, perturbation of Wnt signaling did not have an overall effect on the specification of diencephalic fates, but selectively affected the number of dopaminergic (DA) neurons. We identified the DA progenitor zone in the diencephalic anlage of the neural plate using a two-photon-based uncaging method and showed that the number of non-DA neurons derived from this progenitor zone is not altered by Wnt attenuation. Using birthdating analysis, we determined the timing of the last cell division of DA progenitors and revealed that the change in DA cell number following Wnt inhibition is not due to changes in cell cycle exit kinetics. Conditional inhibition of Wnt and of cell proliferation demonstrated that Wnt restricts the number of DA progenitors during a window of plasticity, which occurs at primary neurogenesis. Finally, we demonstrated that Wnt8b is a modulator of DA cell number that acts through the Fz8a (Fzd8a) receptor and its downstream effector Lef1, and which requires the activity of the Fezl (Fezf2) transcription factor for this process. Our data show that the differential response of distinct neuronal populations to the Wnt signal is not a simple interpretation of their relative anteroposterior position. This study also shows, for the first time, that diencephalic DA population size is modulated inside the neural plate much earlier than expected, concomitant with Wnt-mediated regional patterning events.
Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal ...secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.
The transcriptional coactivator PGC-1α is a key regulator of cellular energy expenditure in peripheral tissues. Recent studies report that PGC-1α-null mice develop late-onset obesity and that the ...neuronal inactivation of PGC-1α causes increased food intake. However, the exact role of PGC-1α in the CNS remains unclear. Here we show that PGC-1α directly regulates the expression of the hypothalamic neuropeptide oxytocin, a known central regulator of appetite. We developed a unique genetic approach in the zebrafish, allowing us to monitor and manipulate PGC-1α activity in oxytocinergic neurons. We found that PGC-1α is coexpressed with oxytocin in the zebrafish hypothalamus. Targeted knockdown of the zebrafish PGC-1α gene activity caused a marked decrease in oxytocin mRNA levels and inhibited the expression of a transgenic GFP reporter driven by the oxytocin promoter. The effect of PGC-1α loss of function on oxytocin gene activity was rescued by tissue-specific re-expression of either PGC-1α or oxytocin precursor in zebrafish oxytocinergic neurons. PGC-1α activated the oxytocin promoter in a heterologous cell culture system, and overexpression of PGC-1α induced ectopic expression of oxytocin in muscles and neurons. Finally, PGC-1α forms an in vivo complex with the oxytocin promoter in fed but not fasted animals. These findings demonstrate that PGC-1α is both necessary and sufficient for the production of oxytocin, implicating hypothalamic PGC-1α in the direct activation of a hypothalamic hormone known to control energy intake.
Summary
The neurohypophysis is a neurovascular interface through which the brain regulates peripheral organs to maintain homeostasis. The molecular mechanisms underlying its formation are poorly ...understood, although the emergence of new genetic and imaging tools has begun to yield new insights. In a recent study, researchers discovered that, in embryonic zebrafish, oxytocin secreted from hypophyseal axons serves as a local angiogenic cue that pulls in nearby blood vessels.
Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small ...molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.
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•Compounds impacting particular genes’ function are highly sought•Most chemicals and overexpressed genes impact cell morphology in the Cell Painting assay•Matching these image profiles can find chemicals that impact a particular gene’s function•This virtual screen using public data found new chemical regulators of several pathways
If a chemical compound and a gene overexpression yield the same cell morphology in the microscopy-based assay Cell Painting, then they are likely to impact the same functions. This principle is exploited to retrieve useful compounds for particular query genes in public Cell Painting datasets.
The hypothalamo-neurohypophyseal system (HNS) is a central point of interface between the hormonal, neuronal and vascular systems and constitutes a conduit through which the brain exerts control over ...peripheral organs. The neuropeptides arginine-vasopressin and oxytocin, highly conserved in all vertebrates, are produced in hypothalamic neurons and released from their axons onto fenestrated capillaries in the neurohypophysis, where they enter the general circulation. In an effort to identify signaling events controlling neurohypopheseal development, we have generated a transgenic oxytocin reporter zebrafish line in which oxytocinergic neurons and their axonal termini are fluorescently labeled, and used it to trace the organization of the HNS during embryogenesis. In particular, we have characterized the interactions between oxytocinergic neurosecretory termini and the developing hypophyseal vasculature, and discovered that locally secreted oxytocin in the neurohypophysis acts as an angiogenic signal required for formation of the hypophyseal vasculature. This project has provided a novel experimental instrument to study the development, assembly and function of the vertebrate HNS, and has uncovered a new molecular mechanism underlying the interaction between neurons and blood vessels during formation of this vital system.
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