Hypoxia-induced proliferation of neural stem cells has a crucial role in brain development. In the brain of Drosophila melanogaster , the optic lobe exhibits progressive hypoxia during larval development. Here, we investigate an alternative oxygen-sensing mechanism within this brain compartment, distinct from the canonical hypoxia signaling pathway mediated by HIF. Using genetic tools, immunostaining, and confocal microscopy, we demonstrate that the loss of the atypical soluble guanylyl cyclase (asGC) subunit Gyc88E , or the ectopic expression of Gyc89Db in neural stem cells leads to increased optic lobe volume. We propose the existence of a link between cGMP signaling and neurogenesis in the developing brain.

CUTie2 is a FRET-based cGMP biosensor tested so far only in cells. To expand its use to multicellular organisms we generated two transgenic Drosophila melanogaster strains that express the biosensor in a tissue-dependent manner. CUTie2 expression and subcellular localization was verified by confocal microscopy. The performance of CUTie2 was analyzed on dissected larval brains by hyperspectral microscopy and flow cytometry. Both approaches confirmed its responsivity, and the latter showed a rapid and reversible change in the fluorescence of the FRET acceptor upon cGMP treatment. This validated reporter system may prove valuable for studying cGMP signaling at organismal level.

In Drosophila, neurons and glial cells derive from neural precursor cells called neuroblasts glioblasts or neuroglioblasts. These neural precursor cells reveal many characteristics reminiscent of neural stem cells that we find in the mammalian central nervous system. We will learn how neuroblasts are generated and specified in a spatio-temporal manner in the developing Drosophila embryo. The relevance of symmetric versus asymmetric stem cell divisions will be conveyed and the major genetic pathway controlling embryonic neurogenesis will be introduced.

In this chapter, we focus on observations that were made in the field of comparative nervous system development by studies carried out in Drosophila and in vertebrate model systems. We will first look at homologous genes that are expressed along the dorsoventral body axis during early neural induction and patterning. Secondly, we discuss findings about the expression and functions of genes involved in anteroposterior patterning of the central nervous system. Despite the significant morphological and developmental differences between insects and vertebrates, there is astonishing conservation between the genetic and molecular mechanisms that control the regionalization of the nervous system. Hence, the data imply that comparable mechanisms operate during embryonic brain development in protostome and deuterostome lineages.

The mammalian brain is probably the most fascinating and complex organ that has evolved over millions of years. In this chapter, we learn about some key genetic factors that control mammalian brain development with a focus on the cerebral cortex. Selected topics highlight oscillatory expression of neurogenic and proneural factors, the relationship between cell cycle control and cell fate and the spatiotemporal generation of neurons in the layered cortex. In a second part, we introduce the neural stem and progenitor cell types in the mammalian neocortex that potentially are the key recent inventions to distinguish higher evolved gyrencephalic from more primitive lissencephalic brains. We discuss the concepts and cellular mechanisms that might have led to neocortex expansion during evolution toward the primate brain.

During development organs and tissues grow in size. This growth can be achieved either by increasing the size of individual cells or by increasing the number of cells by cell proliferation. In an organ like the brain, it is crucial to control the number of cells in order to build functional neuronal circuits. In this chapter, we will look at different types of neural precursor cells in the growing Drosophila larval brain. Each type of neural precursor cell type in Drosophila displays a rather stereotype division pattern. Different modes of division serve to either expand or to differentiate the precursor cell pool. We will learn that different division modes will lead to vastly different outcomes in terms of the generated cell lineage size. The different neural precursor types use also different timings and cellular mechanisms to terminate proliferation once the correct number of progeny cells is reached.

This first chapter provides an overview of the field of neurogenetics including:

Environmental factors such as the availability of oxygen are instructive cues that regulate stem cell maintenance and differentiation. We used a genetically encoded biosensor to monitor the hypoxic state of neural cells in the larval brain of Drosophila. The biosensor reveals brain compartment and cell-type specific levels of hypoxia. The values correlate with differential tracheolation that is observed throughout development between the central brain and the optic lobe. Neural stem cells in both compartments show the strongest hypoxia response while intermediate progenitors, neurons and glial cells reveal weaker responses. We demonstrate that the distance between a cell and the next closest tracheole is a good predictor of the hypoxic state of that cell. Our study indicates that oxygen availability appears to be the major factor controlling the hypoxia response in the developing Drosophila brain and that cell intrinsic and cell-type specific factors contribute to modulate the response in an unexpected manner. This article has an associated First Person interview with the first author of the paper.

Mitotic divisions depend on the timely assembly and proper orientation of the mitotic spindle. Malfunctioning of these processes can considerably delay mitosis, thereby compromising tissue growth and homeostasis, and leading to chromosomal instability. Loss of functional Mms19 drastically affects the growth and development of mitotic tissues in Drosophila larvae and we now demonstrate that Mms19 is an important factor that promotes spindle and astral microtubule (MT) growth, and MT stability and bundling. Mms19 function is needed for the coordination of mitotic events and for the rapid progression through mitosis that is characteristic of neural stem cells. Surprisingly, Mms19 performs its mitotic activities through two different pathways. By stimulating the mitotic kinase cascade, it triggers the localization of the MT regulatory complex TACC/Msps (Transforming Acidic Coiled Coil/Minispindles, the homolog of human ch-TOG) to the centrosome. This activity of Mms19 can be rescued by stimulating the mitotic kinase cascade. However, other aspects of the Mms19 phenotypes cannot be rescued in this way, pointing to an additional mechanism of Mms19 action. We provide evidence that Mms19 binds directly to MTs and that this stimulates MT stability and bundling.

Mitotic divisions depend on the timely assembly and proper orientation of the mitotic spindle. Malfunctioning of these processes can considerably delay mitosis, thereby compromising tissue growth and homeostasis, and leading to chromosomal instability. Here we identified Mms19 as an important player in these processes as it promotes spindle and astral microtubule (MT) growth and consequently regulates spindle orientation and mitosis duration in Drosophila neural stem cells. Loss of functional Mms19 drastically affects the growth and development of mitotic tissues in Drosophila larvae. We found that Mms19 performs its mitotic activities through two different pathways. By stimulating the mitotic kinase cascade, it triggers the localization of the MT regulatory complex TACC/Msps (Transforming Acidic Coiled Coil/Minispindles, the homolog of human ch-TOG) to the centrosome. In addition, we present evidence that Mms19 stimulates MT stability and bundling by binding directly to MTs.

After a peripheral nerve lesion, distal ends of injured axons disintegrate into small fragments that are subsequently cleared by Schwann cells and later by macrophages. Axonal debris clearing is an early step of the repair process that facilitates regeneration. We show here that Schwann cells promote distal cut axon disintegration for timely clearing. By combining cell-based and in vivo models of nerve lesion with mouse genetics, we show that this mechanism is induced by distal cut axons, which signal to Schwann cells through PlGF mediating the activation and upregulation of VEGFR1 in Schwann cells. In turn, VEGFR1 activates Pak1, leading to the formation of constricting actomyosin spheres along unfragmented distal cut axons to mediate their disintegration. Interestingly, oligodendrocytes can acquire a similar behavior as Schwann cells by enforced expression of VEGFR1. These results thus identify controllable molecular cues of a neuron-glia crosstalk essential for timely clearing of damaged axons.

Live cell imaging gives valuable insights into the dynamic biological processes within and between cells. An important aspect of live cell imaging is to keep the cells under best physiological condition and to prevent abnormal cellular behavior, which might be caused by phototoxicity during microscopy. In this chapter we describe a protocol to visualize division patterns of neural stem cells in live whole mount brains of Drosophila larvae. We also present a newly developed live cell chamber that allows us to control the environmental air during live cell imaging. The protocol can be adapted to look at a wide range of cellular and tissue behavior in the Drosophila model system.

Lasting changes in gene expression are critical for the formation of long-term memories (LTMs), depending on the conserved CrebB transcriptional activator. While requirement of distinct neurons in defined circuits for different learning and memory phases have been studied in detail, only little is known regarding the gene regulatory changes that occur within these neurons. We here use the fruit fly as powerful model system to study the neural circuits of CrebB-dependent appetitive olfactory LTM. We edited the CrebB locus to create a GFP-tagged CrebB conditional knockout allele, allowing us to generate mutant, post-mitotic neurons with high spatial and temporal precision. Investigating CrebB-dependence within the mushroom body (MB) circuit we show that MB α/β and α'/β' neurons as well as MBON α3, but not in dopaminergic neurons require CrebB for LTM. Thus, transcriptional memory traces occur in different neurons within the same neural circuit.

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. Here, we characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define PR subtype identity. Our model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.

BACKGROUND:Neural stem cells generate all of the neurons and glial cells in the central nervous system, both during development and in the adult to maintain homeostasis. In the Drosophila optic lobe, neuroepithelial cells progress through two transient progenitor states, PI and PII, before transforming into neuroblasts. Here we analyse the role of Notch signalling in the transition from neuroepithelial cells to neuroblasts. RESULTS:We observed dynamic regulation of Notch signalling: strong activity in PI progenitors, low signalling in PII progenitors, and increased activity after neuroblast transformation. Ectopic expression of the Notch ligand Delta induced the formation of ectopic PI progenitors. Interestingly, we show that the E3 ubiquitin ligase, Neuralized, regulates Delta levels and Notch signalling activity at the transition zone. We demonstrate that the proneural transcription factor, Lethal of scute, is essential to induce expression of Neuralized and promote the transition from the PI progenitor to the PII progenitor state. CONCLUSIONS:Our results show dynamic regulation of Notch signalling activity in the transition from neuroepithelial cells to neuroblasts. We propose a model in which Lethal of scute activates Notch signalling in a non-cell autonomous manner by regulating the expression of Neuralized, thereby promoting the progression between different neural stem cell states.

Cellular processes in the brain allow for most complex computations, which in turn provide the basis of any form of behaviour. With the advent of diffraction-unlimited imaging techniques, it is now possible to study processes in neurons with unprecedented subcellular resolution. Based on the illumination and recording techniques, two approaches are dominating super-resolution microscopy: stimulated emission depletion (STED) microscopy, which is based on image scanning, and stochastic optical reconstruction microscopy (STORM)/photoactivated localization microscopy (PALM), which makes use of single-molecule localization. In the last decade, both approaches led to spectacular insight into the nanostructure of various neuronal compartments. We highlight a selection of studies, in which neuronal structures and processes were imaged with super-resolution, including the cytoskeleton of axons, plasticity in dendritic spines as well as molecular arrangements in presynaptic terminals and active zones.