Puckered and JNK signaling in pioneer neurons coordinates the motor activity of the Drosophila embryo.

Central nervous system organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyze the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling in a subset of pioneer neurons, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating JNK signaling activity in pioneer neurons during early embryogenesis directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization.

JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos.

Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), we observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, our data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization.

JNK signaling and integrins cooperate to maintain cell adhesion during epithelial fusion in Drosophila.

The fusion of epithelial sheets is an essential and conserved morphogenetic event that requires the maintenance of tissue continuity. This is secured by membrane-bound or diffusible signals that instruct the epithelial cells, in a coordinated fashion, to change shapes and adhesive properties and when, how and where to move. Here we show that during Dorsal Closure (DC) in Drosophila, the Jun kinase (JNK) signaling pathway modulates integrins expression and ensures tissue endurance. An excess of JNK activity, as an outcome of a failure in the negative feedback implemented by the dual-specificity phosphatase Puckered (Puc), promotes the loss of integrins [the ß-subunit Myospheroid (Mys)] and amnioserosa detachment. Likewise, integrins signal back to the pathway to regulate the duration and strength of JNK activity. Mys is necessary for the regulation of JNK activity levels and in its absence, puc expression is downregulated and JNK activity increases.

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions.

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

Measuring ventral nerve cord stiffness in live flat-dissected Drosophila embryos by atomic force microscopy.

Drosophila is an amenable system for addressing the mechanics of morphogenesis. We describe a workflow for characterizing the mechanical properties of its ventral nerve cord (VNC), at different developmental stages, in live, flat-dissected embryos employing atomic force microscopy (AFM). AFM is performed with spherical probes, and stiffness (Young's modulus) is calculated by fitting force curves with Hertz's contact model. For complete details on the use and execution of this protocol, please refer to Karkali et al. (2022).

Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase.

For developmental processes, we know most of the gene networks controlling specific cell responses. We still have to determine how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, we still know little about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. We have performed a promoter analysis, searching for potential regulatory sequences of puckered (puc) and identified different specific enhancers directing gene expression in different tissues and at different developmental times. Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila.

Automated Macro Approach to Remove Vitelline Membrane Autofluorescence in Drosophila Embryo 4D Movies.

This chapter provides an ImageJ/Fiji automated macro approach to remove the vitelline membrane autofluorescence in live Drosophila embryo movies acquired in a 4D (3D plus time) fashion. The procedure consists in a segmentation pipeline that can cope with different relative intensities of the vitelline membrane autofluorescence, followed by a developed algorithm that adjusts the extracted outline selection to the shape deformations that naturally occur during Drosophila embryo development. Finally, the fitted selection is used to clear the external glowing halo that, otherwise, would obscure the visualization of the internal embryo labeling upon projection or 3D rendering.

Mechanosensing in the Drosophila nervous system.

Neurons allocated to sense organs respond rapidly to mechanical signals dictating behavioral responses at the organism level. The receptors that transduce these signals, and underlie these senses, are mechanically gated channels. Research on mechanosensation over the past decade, employing in many cases Drosophila as a model, has focused in typifying these receptors and in exploring the different ways, depending on context, in which these mechanosensors are modulated. In this review, we discuss first what we have learned from Drosophila on these mechanisms and we describe the different mechanosensory organs present in the Drosophila larvae and adult. Secondly, we focus on the progress obtained by studying the fly on the characterization of the mechanosensory crosstalk underlying complex behaviors like motor coordination. Finally, turning to a cellular level, we summarize what is known on the mechanical properties and sensing capabilities of neural cells and how they may affect neural physiology and pathology.

Live imaging in Drosophila: The optical and genetic toolkits.

Biological imaging based on light microscopy comes at the core of the methods that let us understanding morphology and its dynamics in synergy to the spatiotemporal distribution of cellular and molecular activities as the organism develops and becomes functional. Non-linear optical tools and superesolution methodologies are under constant development and their applications to live imaging of whole organisms keep improving as we speak. Genetically coded biosensors, multicolor clonal methods and optogenetics in different organisms and, in particular, in Drosophila follow equivalent paths. We anticipate a brilliant future for live imaging providing the roots for the holistic understanding, rather than for individual parts, of development and function at the whole-organism level.

The Drosophila DUSP puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress.

Dual-Specificity Phosphatases (DUSPs) are enzymes that remove phosphate groups from both phospho-tyrosine and phospho-serine/threonine residues. A subgroup of DUSPs specifically targets Mitogen-Activated Protein Kinases (MAPKs) and has been shown to participate in the regulation of differential cellular responses to the large variety of stimuli conveyed by MAPK-pathways. In Drosophila, Puckered has been identified as a DUSP, exhibiting specificity towards the c-Jun-N-terminal kinase (JNK). Recent studies have signified its role in regulating JNK-dependent processes, including immunity, stress tolerance and longevity. Puckered expression depends on the activation of the JNK pathway whereas it's degradation is mediated by the ubiquitin-proteasome system. In this study we show that Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress and that phosphorylation affects the interaction between Puckered and these MAPKs. In silico analysis of the Puckered amino acid sequence revealed several MAPK consensus phosphorylation motifs. Expression of Puckered in the heterologous system of HEK293 cells and subsequent stimulation with arsenite resulted in reduced mobility of Puckered in SDS-PAGE. Similar results were obtained when Puckered was co-expressed with the constitutively active forms of JNK and p38. This mobility shift was abolished by lambda-phosphatase treatment or by simultaneous inhibition of JNK and p38. Analysis by mass-spectrometry identified Puckered phosphorylation in Ser413, though phosphorylation on this site was found irrespective of stimulation. Finally, phosphorylation of Puckered enhanced its interaction both with JNK and p38. Our results suggest a possible functional role of Puckered phosphorylation by MAPKs.