The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.

Dopamine (DA) is synonymous with reward and motivation in mammals1,2. However, only recently has dopamine been linked to motivated behavior and rewarding reinforcement in fruit flies3,4. Instead octopamine (OA) has historically been considered the signal for reward in insects5–7. Here we show using temporal control of neural function in Drosophila that only short-term appetitive memory is reinforced by OA. Moreover, OA-dependent memory formation requires signaling through DA neurons. Part of the OA signal requires the α-adrenergic like OAMB receptor in an identified subset of mushroom body (MB)-targeted DA neurons. OA triggers an increase in intracellular calcium in these DA neurons and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking OA. Analysis of the β-adrenergic like Octβ2R receptor reveals that OA-dependent reinforcement also requires an interaction with DA neurons that control appetitive motivation. These data suggest that sweet taste engages a distributed OA signal that reinforces memory through discrete subsets of MB-targeted DA neurons. In addition, they reconcile prior findings with OA and DA and suggest that reinforcement systems in flies are more similar to mammals than previously envisaged.

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the mechanism by which these mutants cause motor neuron degeneration is not known. We report a novel ALS truncation mutant (R495X) that leads to a relatively severe ALS clinical phenotype compared with FUS missense mutations. Expression of R495X FUS, which abrogates a putative nuclear localization signal at the C-terminus of FUS, in HEK-293 cells and in the zebrafish spinal cord caused a striking cytoplasmic accumulation of the protein to a greater extent than that observed for recessive (H517Q) and dominant (R521G) missense mutants. Furthermore, in response to oxidative stress or heat shock conditions in cultures and in vivo, the ALS-linked FUS mutants, but not wild-type FUS, assembled into perinuclear stress granules in proportion to their cytoplasmic expression levels. These findings demonstrate a potential link between FUS mutations and cellular pathways involved in stress responses that may be relevant to altered motor neuron homeostasis in ALS.

SummaryDopaminergic neurons provide reward learning signals in mammals and insects [1–4]. Recent work in Drosophila has demonstrated that water-reinforcing dopaminergic neurons are different to those for nutritious sugars [5]. Here, we tested whether the sweet taste and nutrient properties of sugar reinforcement further subdivide the fly reward system. We found that dopaminergic neurons expressing the OAMB octopamine receptor [6] specifically convey the short-term reinforcing effects of sweet taste [4]. These dopaminergic neurons project to the β′2 and γ4 regions of the mushroom body lobes. In contrast, nutrient-dependent long-term memory requires different dopaminergic neurons that project to the γ5b regions, and it can be artificially reinforced by those projecting to the β lobe and adjacent α1 region. Surprisingly, whereas artificial implantation and expression of short-term memory occur in satiated flies, formation and expression of artificial long-term memory require flies to be hungry. These studies suggest that short-term and long-term sugar memories have different physiological constraints. They also demonstrate further functional heterogeneity within the rewarding dopaminergic neuron population.

Summary Labile memory is thought to be held in the brain as persistent neural network activity [1–4]. However, it is not known how biologically relevant memory circuits are organized and operate. Labile and persistent appetitive memory in Drosophila requires output after training from the α′β′ subset of mushroom body (MB) neurons and from a pair of modulatory Dorsal Paired Medial (DPM) neurons [5–9]. DPM neurons innervate the entire MB lobe region and appear to be pre- and post-synaptic to the MB [7, 8], consistent with a recurrent network model. Here we identify a role after training for synaptic output from the GABAergic Anterior Paired Lateral (APL) neurons [10, 11]. Blocking synaptic output from APL neurons after training disrupts labile memory but does not affect long-term memory. APL neurons contact DPM neurons most densely in the α′β′ lobes although their processes are intertwined and contact throughout all the lobes. Furthermore, APL contacts MB neurons in the α′ lobe but makes little direct contact with those in the distal α lobe. We propose that APL neurons provide widespread inhibition to stabilize and maintain synaptic specificity of a labile memory trace in a recurrent DPM and MB α′β′ neuron circuit.