Generating parallel representations of position and identity in the olfactory system.

In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. Here, we show that separate cVA-processing streams extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, we identify 47 cell types with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts.

Neurodevelopment: Comparative connectomics and the study of circuit assembly.

A new study reveals the effects of specific single cell type perturbations on the assembly and function of a mechanosensory circuit in the Drosophila melanogaster larva with synaptic resolution. This work paves the way for the use of comparative connectomics to understand general principles governing the development of wiring specificity.

TimerQuant: a modelling approach to tandem fluorescent timer design and data interpretation for measuring protein turnover in embryos.

Studies on signalling dynamics in living embryos have been limited by a scarcity of in vivo reporters. Tandem fluorescent protein timers provide a generic method for detecting changes in protein population age and thus provide readouts for signalling events that lead to changes in protein stability or location. When imaged with quantitative dual-colour fluorescence microscopy, tandem timers offer detailed 'snapshot' readouts of signalling activity from subcellular to organismal scales, and therefore have the potential to revolutionise studies in developing embryos. Here we use computer modelling and embryo experiments to explore the behaviour of tandem timers in developing systems. We present a mathematical model of timer kinetics and provide software tools that will allow experimentalists to select the most appropriate timer designs for their biological question, and guide interpretation of the obtained readouts. Through the generation of a series of novel zebrafish reporter lines, we confirm experimentally that our quantitative model can accurately predict different timer responses in developing embryos and explain some less expected findings. For example, increasing the FRET efficiency of a tandem timer actually increases the ability of the timer to detect differences in protein half-life. Finally, while previous studies have used timers to monitor changes in protein turnover, our model shows that timers can also be used to facilitate the monitoring of gene expression kinetics in vivo.

Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.

The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process.

Directional tissue migration through a self-generated chemokine gradient.

The directed migration of cell collectives is a driving force of embryogenesis. The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients. One hypothetical way to free migrating collectives from the requirement of long-range gradients would be through the self-generation of local gradients that travel with them, a strategy that potentially allows self-determined directionality. However, a lack of tools for the visualization of endogenous guidance cues has prevented the demonstration of such self-generated gradients in vivo. Here we define the in vivo dynamics of one key guidance molecule, the chemokine Cxcl12a, by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals. Using the zebrafish lateral line primordium as a model, we show that migrating cell collectives can self-generate gradients of chemokine activity across their length via polarized receptor-mediated internalization. Finally, by engineering an external source of the atypical receptor Cxcr7 that moves with the primordium, we show that a self-generated gradient mechanism is sufficient to direct robust collective migration. This study thus provides, to our knowledge, the first in vivo proof for self-directed tissue migration through local shaping of an extracellular cue and provides a framework for investigating self-directed migration in many other contexts including cancer invasion.