Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper.

Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. Here, we show that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of Notch(OFF) or Notch(ON) neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their Notch(ON) progeny systematically die through Reaper activity, whereas later, their Notch(OFF) progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. We conclude that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype.

Increased sugar valuation contributes to the evolutionary shift in egg-laying behavior of the fruit pest Drosophila suzukii.

Behavior evolution can promote the emergence of agricultural pests by changing their ecological niche. For example, the insect pest Drosophila suzukii has shifted its oviposition (egg-laying) niche from fermented fruits to ripe, non-fermented fruits, causing significant damage to a wide range of fruit crops worldwide. We investigate the chemosensory changes underlying this evolutionary shift and ask whether fruit sugars, which are depleted during fermentation, are important gustatory cues that direct D. suzukii oviposition to sweet, ripe fruits. We show that D. suzukii has expanded its range of oviposition responses to lower sugar concentrations than the model D. melanogaster, which prefers to lay eggs on fermented fruit. The increased response of D. suzukii to sugar correlates with an increase in the value of sugar relative to a fermented strawberry substrate in oviposition decisions. In addition, we show by genetic manipulation of sugar-gustatory receptor neurons (GRNs) that sugar perception is required for D. suzukii to prefer a ripe substrate over a fermented substrate, but not for D. melanogaster to prefer the fermented substrate. Thus, sugar is a major determinant of D. suzukii's choice of complex substrates. Calcium imaging experiments in the brain's primary gustatory center (suboesophageal zone) show that D. suzukii GRNs are not more sensitive to sugar than their D. melanogaster counterparts, suggesting that increased sugar valuation is encoded in downstream circuits of the central nervous system (CNS). Taken together, our data suggest that evolutionary changes in central brain sugar valuation computations are involved in driving D. suzukii's oviposition preference for sweet, ripe fruit.

Differentially timed extracellular signals synchronize pacemaker neuron clocks.

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in Drosophila larvae, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. We also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.

A two-tiered mechanism for stabilization and immobilization of E-cadherin.

Epithelial tissues maintain a robust architecture which is important for their barrier function, but they are also remodelled through the reorganization of cell-cell contacts. Tissue stability requires intercellular adhesion mediated by E-cadherin, in particular its trans-association in homophilic complexes supported by actin filaments through beta- and alpha-catenin. How alpha-catenin dynamic interactions between E-cadherin/beta-catenin and cortical actin control both stability and remodelling of adhesion is unclear. Here we focus on Drosophila homophilic E-cadherin complexes rather than total E-cadherin, including diffusing 'free' E-cadherin, because these complexes are a better proxy for adhesion. We find that E-cadherin complexes partition in very stable microdomains (that is, bona fide adhesive foci which are more stable than remodelling contacts). Furthermore, we find that stability and mobility of these microdomains depend on two actin populations: small, stable actin patches concentrate at homophilic E-cadherin clusters, whereas a rapidly turning over, contractile network constrains their lateral movement by a tethering mechanism. alpha-Catenin controls epithelial architecture mainly through regulation of the mobility of homophilic clusters and it is largely dispensable for their stability. Uncoupling stability and mobility of E-cadherin complexes suggests that stable epithelia may remodel through the regulated mobility of very stable adhesive foci.

Evolution of Ovipositor Length in Drosophila suzukii Is Driven by Enhanced Cell Size Expansion and Anisotropic Tissue Reorganization.

Morphological diversity is dominated by variation in body proportion [1], which can be described with scaling relationships and mathematical equations, following the pioneering work of D'Arcy Thompson [2] and Julian Huxley [3]. Yet, the cellular processes underlying divergence in size and shape of morphological traits between species remain largely unknown [4-8]. Here, we compare the ovipositors of two related species, Drosophila melanogaster and D. suzukii. D. suzukii has switched its egg-laying niche from rotting to ripe fruit [9]. Along with this shift, the D. suzukii ovipositor has undergone a significant change in size and shape [10]. Using an allometric approach, we find that, while adult ovipositor width has hardly changed between the species, D. suzukii ovipositor length is almost double that of D. melanogaster. We show that this difference mostly arises in a 6-h time window during pupal development. We observe that the developing ovipositors of the two species comprise an almost identical number of cells, with a similar profile of cell shapes and orientations. After cell division stops, we find that the ovipositor area continues to grow in both species through the isotropic expansion of cell apical area and the anisotropic cellular reorganization of the tissue. Remarkably, we find that the lengthening of the D. suzukii ovipositor compared to that of D. melanogaster results from the combination of the accelerated expansion of apical cell size and the enhanced anisotropic rearrangement of cells in the tissue. Therefore, the quantitative fine-tuning of morphogenetic processes can drive evolutionary changes in organ size and shape.

Imaging cellular and molecular dynamics in live embryos using fluorescent proteins.

With the live imaging of embryos, the dynamics of developmental processes, such as tissue remodeling, cell morphogenesis, and, campus protein dynamics can be observed and quantified. This has greatly improved the mechanistic understanding of biological processes. Here we describe how embryos can be prepared for imaging mainly, but not only, fluorescent proteins and probes. This chapter is a users' guide that addresses the following aspects of fluorescent embryo imaging: (1) How to handle and prepare embryos for live microscopy. (2) What microscopic setups are available for embryo imaging and what should they be used for. (3) How to practically use fluorescent imaging setups depending on the experimental context: large-scale imaging of multiple embryos, high-resolution four-dimensional imaging of single embryos, studies of protein dynamics, and so on. (4) Finally, we focus on pitfalls and how to overcome a variety of possible problems encountered during live imaging.

Evolution of Multiple Sensory Systems Drives Novel Egg-Laying Behavior in the Fruit Pest Drosophila suzukii.

The rise of a pest species represents a unique opportunity to address how species evolve new behaviors and adapt to novel ecological niches [1]. We address this question by studying the egg-laying behavior of Drosophila suzukii, an invasive agricultural pest species that has spread from Southeast Asia to Europe and North America in the last decade [2]. While most closely related Drosophila species lay their eggs on decaying plant substrates, D. suzukii oviposits on ripening fruit, thereby causing substantial economic losses to the fruit industry [3-8]. D. suzukii has evolved an enlarged, serrated ovipositor that presumably plays a key role by enabling females to pierce the skin of ripe fruit [9]. Here, we explore how D. suzukii selects oviposition sites, and how this behavior differs from that of closely related species. We have combined behavioral experiments in multiple species with neurogenetics and mutant analysis in D. suzukii to show that this species has evolved a specific preference for oviposition on ripe fruit. Our results also establish that changes in mechanosensation, olfaction, and presumably gustation have contributed to this ecological shift. Our observations support a model in which the emergence of D. suzukii as an agricultural pest is the consequence of the progressive modification of several sensory systems, which collectively underlie a radical change in oviposition behavior.

Circadian rhythms in neuronal activity propagate through output circuits.

Twenty-four hour rhythms in behavior are organized by a network of circadian pacemaker neurons. Rhythmic activity in this network is generated by intrinsic rhythms in clock neuron physiology and communication between clock neurons. However, it is poorly understood how the activity of a small number of pacemaker neurons is translated into rhythmic behavior of the whole animal. To understand this, we screened for signals that could identify circadian output circuits in Drosophila melanogaster. We found that leucokinin neuropeptide (LK) and its receptor (LK-R) were required for normal behavioral rhythms. This LK/LK-R circuit connects pacemaker neurons to brain areas that regulate locomotor activity and sleep. Our experiments revealed that pacemaker neurons impose rhythmic activity and excitability on LK- and LK-R-expressing neurons. We also found pacemaker neuron-dependent activity rhythms in a second circadian output pathway controlled by DH44 neuropeptide-expressing neurons. We conclude that rhythmic clock neuron activity propagates to multiple downstream circuits to orchestrate behavioral rhythms.

Molecular bases of cell-cell junctions stability and dynamics.

Epithelial cell-cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell-cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell-cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell-cell junctions.

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation.

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.

Map positions of third chromosomal female sterile and lethal mutations of Drosophila melanogaster.

Chromosomal mutations induced by ethyl methanesulfonate (EMS) treatment can cause female sterility or maternal-effect lethality in Drosophila. EMS is particularly useful to researchers because it creates mutations independent of position effects. However, because researchers have little control over the chromosomal site of mutation, post-mutagenic genetic mapping is required to determine the cytological location of the mutation. To make a valuable set of mutants more useful to the research community, we have mapped the uncharacterized part of the female-sterile - maternal-effect lethal Tubingen collection. We mapped 49 female-sterile - maternal-effect lethal alleles and 72 lethal alleles to individual deficiency intervals on the third chromosome. In addition, we analyzed the phenotype of ovaries resulting from female sterile mutations. The observed phenotypes range from tumorous ovaries and early blocks in oogenesis, to later blocks, slow growth, blocks in stage 10, to apparently full development of the ovary. The mapping and phenotypic characterization of these 121 mutations provide the necessary information for the researcher to consider a specific mutant as a candidate for their gene of interest.