Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila.

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones.

Extensive studies of growing axons have revealed many individual components and protein interactions that guide neuronal morphogenesis. Despite this, however, we lack any clear picture of the emergent mechanism by which this nanometer-scale biochemistry generates the multimicron-scale morphology and cell biology of axon growth and guidance in vivo. To address this, we studied the downstream effects of the Abl signaling pathway using a computer simulation software (MEDYAN) that accounts for mechanochemical dynamics of active polymers. Previous studies implicate two Abl effectors, Arp2/3 and Enabled, in Abl-dependent axon guidance decisions. We now find that Abl alters actin architecture primarily by activating Arp2/3, while Enabled plays a more limited role. Our simulations show that simulations mimicking modest levels of Abl activity bear striking similarity to actin profiles obtained experimentally from live imaging of actin in wild-type axons in vivo. Using a graph theoretical filament-filament contact analysis, moreover, we find that networks mimicking hyperactivity of Abl (enhanced Arp2/3) are fragmented into smaller domains of actin that interact weakly with each other, consistent with the pattern of actin fragmentation observed upon Abl overexpression in vivo. Two perturbative simulations further confirm that high-Arp2/3 actin networks are mechanically disconnected and fail to mount a cohesive response to perturbation. Taken together, these data provide a molecular-level picture of how the large-scale organization of the axonal cytoskeleton arises from the biophysics of actin networks.

NIH BRAIN Circuits Programs: An Experiment in Supporting Team Neuroscience.

The NIH BRAIN Initiative is aimed at revolutionizing our understanding of the human brain. Here, we present a discussion of support for team research in investigative neuroscience at different stages and on various scales.

The promise of the BRAIN initiative: NIH strategies for understanding neural circuit function.

New neurotechnologies fueled by the BRAIN Initiative now allow investigators to map, monitor and modulate complex neural circuits, enabling the pursuit of research questions previously considered unapproachable. Yet it is the convergence of molecular neuroscience with the new systems neuroscience that promises the greatest future advances. This is particularly true for our understanding of nervous system disorders, some of which have known molecular drivers or pathology but result in unknown perturbations in circuit function. NIH-supported research on "BRAIN Circuits" programs integrate experimental, analytic, and theoretical capabilities for analysis of specific neural circuits and their contributions to perceptions, motivations, and actions. In this review, we describe the BRAIN priority areas, review our strategy for balancing early feasibility with mature projects, and the balance of individual with team science for this 'BRAIN Circuits' program. We also highlight the diverse portfolio of techniques, species, and neural systems represented in these projects.

Dynamic morphogenesis of a pioneer axon in Drosophila and its regulation by Abl tyrosine kinase.

The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. We here dissect this process using live imaging of the TSM1 axon of the developing Drosophila wing. We find that the growth cone is almost purely filopodial, and that it extends by a protrusive mode of growth. Quantitative analysis reveals two separate groups of growth cone properties that together account for growth cone structure and dynamics. The core morphological features of the growth cone are strongly correlated with one another and define two discrete morphs. Genetic manipulation of a critical mediator of axon guidance signaling, Abelson (Abl) tyrosine kinase, shows that while Abl weakly modulates the ratio of the two morphs it does not greatly change their properties. Rather, Abl primarily regulates the second group of properties, which report the organization and distribution of actin in the growth cone and are coupled to growth cone velocity. Other experiments dissect the nature of that regulation of actin organization and how it controls the spatial localization of filopodial dynamics and thus axon extension. Together, these observations suggest a novel, probabilistic mechanism by which Abl biases the stochastic fluctuations of growth cone actin to direct axon growth and guidance.

Abl signaling directs growth of a pioneer axon in Drosophila by shaping the intrinsic fluctuations of actin.

The fundamental problem in axon growth and guidance is understanding how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. Live imaging of the TSM1 axon of the developing Drosophila wing has shown that the essential role of the core guidance signaling molecule, Abelson (Abl) tyrosine kinase, is to modulate the organization and spatial localization of actin in the advancing growth cone. Here, we dissect in detail the properties of that actin organization and its consequences for growth cone morphogenesis and motility. We show that advance of the actin mass in the distal axon drives the forward motion of the dynamic filopodial domain that defines the growth cone. We further show that Abl regulates both the width of the actin mass and its internal organization, spatially biasing the intrinsic fluctuations of actin to achieve net advance of the actin, and thus of the dynamic filopodial domain of the growth cone, while maintaining the essential coherence of the actin mass itself. These data suggest a model whereby guidance signaling systematically shapes the intrinsic, stochastic fluctuations of actin in the growth cone to produce axon growth and guidance.

Morphological and molecular characterization of adult midgut compartmentalization in Drosophila.

Although the gut is a central organ of Eumetazoans and is essential for organismal health, our understanding of its morphological and molecular determinants remains rudimentary. Here, we provide a comprehensive atlas of Drosophila adult midgut. Specifically, we uncover a fine-grained regional organization consisting of 14 subregions with distinct morphological, histological, and genetic properties. We also show that Drosophila intestinal regionalization is defined after adult emergence, remains stable throughout life, and reestablishes following acute tissue damage. Additionally, we show that this midgut compartmentalization is achieved through the interplay between pan-midgut and regionalized transcription factors, in concert with spatial activities of morphogens. Interestingly, disruption of the midgut compartmentalization leads to a loss of intestinal homeostasis characterized by an increase in stem cell proliferation and aberrant immune responses. Our integrative analysis of Drosophila midgut compartmentalization provides insights into the conserved mechanisms underlying intestinal regionalization in metazoans.