Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

Proliferation of neural precursors in the optic lobe of Manduca sexta is controlled by circulating steroids and by local production of nitric oxide (NO). Diaphorase staining, anti-NO synthase (NOS) immunocytochemistry and the NO-indicator, DAF-2, show that cells throughout the optic anlage contain NOS and produce NO. Signaling via NO inhibits proliferation in the anlage. When exposed to low levels of ecdysteroid, NO production is stimulated and proliferation ceases. When steroid levels are increased, NO production begins to decrease within 15 minutes independent of RNA or protein synthesis and cells rapidly resume proliferation. Resumption of proliferation is not due simply to the removal of NO repression though, but also requires an ecdysteroid stimulatory pathway. The consequence of these opposing pathways is a sharpening of the responsiveness to the steroid, thereby facilitating a tight coordination between development of the different elements of the adult visual system.

The eye primordium of the moth, Manduca sexta, shows two different developmental responses to ecdysteroids depending on the concentration to which it is exposed. Tonic exposure to moderate levels of 20-hydroxyecdysone (20E) or its precursor, ecdysone, are required for progression of the morphogenetic furrow across the primordium. Proliferation, cell-type specification and organization of immature ommatidial clusters occur in conjunction with furrow progression. These events can be reversibly started or stopped in cultured primordia simply by adjusting levels of ecdysteroid to be above or below a critical threshold concentration. In contrast, high levels of 20E cause maturation of the photoreceptors and the support cells that comprise the ommatidia. Ommatidial maturation normally occurs after the furrow has crossed the primordium, but premature exposure to high levels of 20E at any time causes precocious maturation. In such cases, the furrow arrests irreversibly and cells behind the furrow produce a well-formed, but miniature, eye. Precocious and catastrophic metamorphosis occurs throughout such animals, suggesting that ecdysteroids control development of other tissues in a manner similar to the eye. The threshold concentrations of 20E required for furrow progression versus ommatidial maturation differ by about 17-fold. This capacity to regulate distinct phases of development by different concentrations of a single hormone is probably achieved by differential sensitivity of target gene promoters to induction by the hormone-bound receptor(s).

Loners-individuals out of sync with a coordinated majority-occur frequently in nature. Are loners incidental byproducts of large-scale coordination attempts, or are they part of a mosaic of life-history strategies? Here, we provide empirical evidence of naturally occurring heritable variation in loner behavior in the model social amoeba Dictyostelium discoideum. We propose that Dictyostelium loners-cells that do not join the multicellular life stage-arise from a dynamic population-partitioning process, the result of each cell making a stochastic, signal-based decision. We find evidence that this imperfectly synchronized multicellular development is affected by both abiotic (environmental porosity) and biotic (signaling) factors. Finally, we predict theoretically that when a pair of strains differing in their partitioning behavior coaggregate, cross-signaling impacts slime-mold diversity across spatiotemporal scales. Our findings suggest that loners could be critical to understanding collective and social behaviors, multicellular development, and ecological dynamics in D. discoideum. More broadly, across taxa, imperfect coordination of collective behaviors might be adaptive by enabling diversification of life-history strategies.

The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.

In Neuroscience, the structure of a circuit has often been used to intuit function - an inversion of Louis Kahn's famous dictum, `Form follows function' (Kristan and Katz 2006). However, different brain networks may utilize different network architectures to solve the same problem. The olfactory circuits of two insects, the Locust, and the fruit fly, , serve the same function - to identify and discriminate odors. The neural circuitry that achieves this shows marked structural differences. Projection neurons (PN) in the antennal lobe (AL) innervate Kenyon cells (KC) of the mushroom body (MB). In locust, each KC receives inputs from ∼50% PNs, a scheme that maximizes the difference between inputs to any two of ∼50,000 KCs. In contrast, in drosophila, this number is only 5% and appears sub-optimal. Using a computational model of the olfactory system, we show the activity of KCs is sufficiently high-dimensional that it can separate similar odors regardless of the divergence of PN-KC connections. However, when temporal patterning encodes odor attributes, dense connectivity outperforms sparse connections.Increased separability comes at the cost of reliability. The disadvantage of sparse connectivity can be mitigated by incorporating other aspects of circuit architecture seen in drosophila. Our simulations predict that drosophila and locust circuits lie at different ends of a continuum where the drosophila gives up on the ability to resolve similar odors to generalize across varying environments, while the locust separates odor representations but risks misclassifying noisy variants of the same odor. How does the structure of a network affect its function? We address this question in the context of two olfactory systems that serve the same function, to distinguish the attributes of different odorants, but do so using markedly distinct architectures. In the locust, the probability of connections between projection neurons and Kenyon cells - a layer downstream - is nearly 50%. In contrast, this number is merely 5% in drosophila. We developed computational models of these networks to understand the relative advantages of each connectivity. Our analysis reveals that the two systems exist along a continuum of possibilities that balance two conflicting goals - separating the representations of similar odors while grouping together noisy variants of the same odor.