Developmental genes can have complex cis-regulatory regions, with multiple enhancers scattered across stretches of DNA spanning tens or hundreds of kilobases. Early work revealed remarkable modularity of enhancers, where distinct regions of DNA, bound by combinations of transcription factors, drive gene expression in defined spatio-temporal domains. Nevertheless, a few reports have shown that enhancer function may be required in multiple developmental stages, implying that regulatory elements can be pleiotropic. In these cases, it is not clear whether the pleiotropic enhancers employ the same transcription factor binding sites to drive expression at multiple developmental stages or whether enhancers function as chromatin scaffolds, where independent sets of transcription factor binding sites act at different stages. In this work we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive gene expression in multiple tissues and developmental stages at varying levels of redundancy. We have explored how this pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, whereas for a second enhancer, these roles are largely encoded by distinct transcription factor binding sites. Our data suggest that enhancer pleiotropy might be a common feature of cis-regulatory regions of developmental genes and that this pleiotropy can be encoded through multiple genetic architectures.

Genomics revolutionized the field of social insect research by providing powerful tools to understand the relationship between genes, physiology and behaviour of social insects. Notably, analysis of gene expression and methylation patterns in the different castes of insect colonies highlighted many genes that likely underlie caste-specific physiological and behavioural phenotypes. However, earlier studies of social insect genomes lacked an ‘evolutionary’ context. Out of the millions of DNA bases found in the genome of a social insect, which pieces were most important to fitness over the timescale of social evolution? Here, we review a burgeoning body of literature that utilizes between-species or within-species genomic comparisons to highlight the evolutionary forces that have shaped social insect genomes. These pioneering phylogenetic and population genomic studies provide a critically needed evolutionary context to social insect genomes and underscore the importance of adaptive changes in physiology and behaviour in social evolution.

Quantitative analysis of neuron morphology is essential in order to develop our understanding of circuit organisation and development. The recent acquisition of whole-brain electron microscopy-based (EM) reconstructions of the Drosophila melanogaster nervous system now provide the resolution needed to examine morphology at scale. Utilising these data, together with new computational tools, we extract and analyse the dendrites of all T4 and T5 neurons within one hemisphere (n \~ 6000).T4 and T5 neurons are the first uniquely direction-selective neurons in the visual pathway, and are classified into four subtypes (a,b,c, and d). Each subtype encodes one of four cardinal motion directions (up, down, forwards, backwards). The dendrites of these neurons form in two distinct neuropils, the Medulla (T4) and the Lobula (T5), and are asymmetrically oriented in a direction inverse to the direction of motion which they encode. However, their densely overlapping and compact arbours has made rigorous morphological quantification challenging. The presence of differences beyond their characteristic orientation, both between T4 and T5, as well as within subtypes, has remained poorly understood.Our analysis reveals a high degree of structural similarity across both types and subtypes. Particularly, measures of geometry and graph topology show only minor variation, with no consistent separation between T4 and T5, or their subtypes.These results indicate that, despite forming in different neuropils, and serving distinct motion directions, T4 and T5 dendrites follow closely aligned morphological patterns. This suggests that their arborization may be governed by shared developmental constraints and mechanisms.

Asthma is a common debilitating inflammatory lung disease affecting over 200 million people worldwide. Here, we investigated neurogenic components involved in asthmatic-like attacks using the ovalbumin-sensitized murine model of the disease, and identified a specific population of neurons that are required for airway hyperreactivity. We show that ablating or genetically silencing these neurons abolished the hyperreactive broncho-constrictions, even in the presence of a fully developed lung inflammatory immune response. These neurons are found in the vagal ganglia and are characterized by the expression of the transient receptor potential vanilloid 1 (TRPV1) ion channel. However, the TRPV1 channel itself is not required for the asthmatic-like hyperreactive airway response. We also demonstrate that optogenetic stimulation of this population of TRP-expressing cells with channelrhodopsin dramatically exacerbates airway hyperreactivity of inflamed airways. Notably, these cells express the sphingosine-1-phosphate receptor 3 (S1PR3), and stimulation with a S1PR3 agonist efficiently induced broncho-constrictions, even in the absence of ovalbumin sensitization and inflammation. Our results show that the airway hyperreactivity phenotype can be physiologically dissociated from the immune component, and provide a platform for devising therapeutic approaches to asthma that target these pathways separately.

The palette of tools for stimulation and regulation of neural activity is continually expanding. One of the new methods being introduced is magnetogenetics, where mechano-sensitive and thermo-sensitive ion channels are genetically engineered to be closely coupled to the iron-storage protein ferritin. Such genetic constructs could provide a powerful new way of non-invasively activating ion channels in-vivo using external magnetic fields that easily penetrate biological tissue. Initial reports that introduced this new technology have sparked a vigorous debate on the plausibility of physical mechanisms of ion channel activation by means of external magnetic fields. I argue that the initial criticisms leveled against magnetogenetics as being physically implausible were possibly based on the overly simplistic and unnecessarily pessimistic assumptions about the magnetic spin configurations of iron in ferritin protein. Additionally, all the possible magnetic-field-based mechanisms of ion channel activation in magnetogenetics might not have been fully considered. I present and propose several new magneto-mechanical and magneto-thermal mechanisms of ion channel activation by iron-loaded ferritin protein that may elucidate and clarify some of the mysteries that presently challenge our understanding of the reported biological experiments. Finally, I present some additional puzzles that will require further theoretical and experimental investigation.

Serial section Microscopy is an established method for volumetric anatomy reconstruction. Section series imaged with Electron Microscopy are currently vital for the reconstruction of the synaptic connectivity of entire animal brains such as that of Drosophila melanogaster. The process of removing ultrathin layers from a solid block containing the specimen, however, is a fragile procedure and has limited precision with respect to section thickness. We have developed a method to estimate the relative z-position of each individual section as a function of signal change across the section series. First experiments show promising results on both serial section Transmission Electron Microscopy (ssTEM) data and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) series. We made our solution available as Open Source plugins for the TrakEM2 software and the ImageJ distribution Fiji.

Serial section Microscopy is an established method for volumetric anatomy reconstruction. Section series imaged with Electron Microscopy are currently vital for the reconstruction of the synaptic connectivity of entire animal brains such as that of Drosophila melanogaster. The process of removing ultrathin layers from a solid block containing the specimen, however, is a fragile procedure and has limited precision with respect to section thickness. We have developed a method to estimate the relative z-position of each individual section as a function of signal change across the section series. First experiments show promising results on both serial section Transmission Electron Microscopy (ssTEM) data and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) series. We made our solution available as Open Source plugins for the TrakEM2 software and the ImageJ distribution Fiji.

During larval life most of the thoracic neuroblasts (NBs) in Drosophila undergo a second phase of neurogenesis to generate adult-specific neurons that remain in an immature, developmentally stalled state until pupation. Using a combination of MARCM and immunostaining with a neurotactin antibody Truman et al. (2004) identified 24 adult specific NB lineages within each thoracic hemineuromere of the larval ventral nervous system (VNS) but because the neurotactin labeling of lineage tracts disappearing early in metamorphosis they were unable extend the identification of the these lineages into the adult. Here we show that immunostaining with an antibody against the cell adhesion molecule Neuroglian reveals the same larval secondary lineage projections through metamorphosis and by identifying each neuroglian positive tract at selected stages we have traced the larval hemilineage tracts for all three thoracic neuromeres through metamorphosis into the adult. To validate tract identifications we used the genetic toolkit developed by Harris et al. (2015) to preserve hemilineage specific GAL4 expression patterns from larval into the adult stage. The immortalized expression proved a powerful confirmation of the analysis of the neuroglian scaffold. This work has enabled us to directly link the secondary, larval NB lineages to their adult counterparts. The data provide an anatomical framework that 1) makes it possible to assign most neurons to their parent lineage and 2) allows more precise definitions of the neuronal organization of the adult VNS based in developmental units/rules. This article is protected by copyright. All rights reserved.

Active dendritic synaptic integration enhances the computational power of neurons. Such nonlinear processing generates an object-localization signal in the apical dendritic tuft of layer 5B cortical pyramidal neurons during sensory-motor behavior. Here, we employ electrophysiological and optical approaches in brain slices and behaving animals to investigate how excitatory synaptic input to this distal dendritic compartment influences neuronal output. We find that active dendritic integration throughout the apical dendritic tuft is highly compartmentalized by voltage-gated potassium (KV) channels. A high density of both transient and sustained KV channels was observed in all apical dendritic compartments. These channels potently regulated the interaction between apical dendritic tuft, trunk, and axosomatic integration zones to control neuronal output in vitro as well as the engagement of dendritic nonlinear processing in vivo during sensory-motor behavior. Thus, KV channels dynamically tune the interaction between active dendritic integration compartments in layer 5B pyramidal neurons to shape behaviorally relevant neuronal computations.

Fluorescence microscopy has evolved from a purely observational tool to a platform for quantitative, hypothesis-driven research. As such, the demand for faster and less phototoxic imaging modalities has spurred a rapid growth in light sheet fluorescence microscopy (LSFM). By restricting the excitation to a thin plane, LSFM reduces the overall light dose to a specimen while simultaneously improving image contrast. However, the defining characteristics of light sheet microscopes subsequently warrant unique considerations in their use for quantitative experiments. In this Perspective, we outline many of the pitfalls in LSFM that can compromise analysis and confound interpretation. Moreover, we offer guidance in addressing these caveats when possible. In doing so, we hope to provide a useful resource for life scientists seeking to adopt LSFM to quantitatively address complex biological hypotheses.