Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

Due to its genetic manipulability and relatively short reproductive cycle, genetic screens are often carried out in the fruit fly, Drosophila melanogaster. Deficiency “kits” that cover the Drosophila genome with a minimum number of lines have been established by other groups to facilitate gene mapping. These kits cannot be systematically analyzed for many phenotypes, however, because embryos homozygous for many deficiencies fail to develop due to the loss of key gene products. To create new kits that can be screened for more phenotypes, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ~400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. Screens of this kit can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16 (thus allowing for central nervous system development), covers ~50% of the genome. We have screened this smaller kit for motor axon guidance phenotypes, and we present examples of new axon guidance phenotypes in the central nervous system and neuromuscular system. Through screening of these kits, we also found deficiencies that fail to stain with monoclonal antibody BP102, which recognizes an unknown epitope on the proximal segments of central nervous system axons. In addition, we have found a deficiency that exhibits ectopic BP102 staining on peripheral sensory neurons. By defining the single genes under these deficiencies, we have obtained evidence that BP102 may recognize a chondroitin sulfate proteoglycan and that BP102 epitope expression is regulated by matrix metalloproteinase 1. Thus, in addition to this screen providing information about motor axon guidance in the embryo, we have also been able to further characterize an antibody that is frequently used by the Drosophila community.

Prenatal brain development is mainly accomplished by extensive proliferation of neuronal precursor cells whereas postnatal brain growth in mammals is mainly mediated by the growth of those post-mitotic nerve cells. The neuron size and the branching pattern of the dendritic tree are highly controlled during development to enable the proper connectivity of neuronal circuits and the accurate electrical transmission in the adult which is a prerequisite for the brain to function normally. Aberrations in size, morphology or connectivity have been shown to be the cause for various brain disorders. Neuron size and dendrite development are controlled by intrinsic mechanisms, trophic factors and neuronal activity, processes that need the concerted action of a plethora of signaling molecules. A central integrator of various signaling cascades is the mammalian target of rapamycin (mTOR) and as such it contributes to brain development and function and is thus also implicated in the pathophysiology of psychiatric disorders. mTOR is a serine threonine protein kinase that is highly conserved from yeast to humans and has been found to be part of at least two multi-protein complexes mTORC1 and mTORC2. The formation of mTORC1 is dependent on the protein raptor whereas mTORC2 assembly relies on the protein rictor. In recent years a complex picture about the function of mTORC1 has emerged by use of rapamycin, an immunosuppressive drug that acutely inhibits mTORC1 formation and activity and has attributed mTORC1 a major role in the regulation of cell size and proliferation. However, because the activity of mTORC2 is only depleted upon long term application of rapamycin, research advancement on its function was thus far impeded. Due to the early embryonic lethality of raptor or rictor knockout in mammals conditional knockout models were constructed. Whereas tissue specific knockout of raptor led to characteristic alterations, knockout of rictor in several organs such as skeletal muscle and adipose tissue provided none or only a weak phenotype. Several cell culture studies assigned mTORC2 a role in cytoskeletal modifications but in vivo confirmation is still lacking. The current knowledge about mTORC2 is restricted to the downstream targets Akt/PKB (proteinkinase B) and PKC (protein kinase C) which belong to the AGC kinase family. Those kinases are reported to influence cell morphology, growth and survival and are also essential regulators of brain development and function. PKCs are involved in synaptic plasticity and neurotransmitter release and, hence, also in the pathophysiological mechanisms of psychiatric disorders especially in schizophrenia and bipolar disorder. Concordantly, several psychiatric agents have been shown to alter PKC signaling. This emphasizes the urge to analyze the role of mTORC2 in the central nervous system. In this dissertation the role of mTORC2 was analyzed in the central nervous system and in specific sub-populations of neurons by deletion of rictor. I discovered, that in contrast to all other organs analyzed so far, rictor knockout in the brain reveals a pronounced phenotype. The brain-size of those mice shows an enormous reduction to almost half of that of control mice which is caused mainly by the reduction of neuron size. The reduced cell size is observed in neurons derived from different brain areas in vitro and in vivo but is most prominent in Purkinje cells of the cerebellum, the cell type with highest rictor expression. In addition, dendrite morphology is majorly disrupted and the formation of dendritic spines is affected which correlates with a decreased neuronal activity. The Purkinje cell phenotype can also be reproduced in a Purkinje cell specific knockout of rictor and thus demonstrates that the effect of rictor deletion in neurons is cell autonomous. Moreover, Purkinje cell axonal path-finding is affected which correlates with the decrease in phosphorylation of the neuron specific PKC target protein GAP-43, a known regulator for axon growth and path-finding. Molecular analysis reveals that rictor is essential for the activity of all conventional PKC isoforms and the novel PKCe in vivo and in vitro in neurons which influences the function of downstream targets important for cytoskeleton modifications such as GAP-43, MARCKs and neurofascin. In addition, rictor controls the phosphorylation of Akt but does not alter mTORC1 signaling towards its downstream effectors. In summary it becomes clear that rictor is important in the development and maturation of neurons and controls their size and neuron structure which influences the entire brain function and affects the behavior of the mice. Thus, these data encompass a new role of rictor in CNS disorders.

i.Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challengingin vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP1-10/11) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP1-10/11system in fruit flies. This approach depends on the reconstitution of the GFP1-10and GFP11fragments, creating a fluorescence signal. We insert theGFP11fragment into a specific genomic locus while expressing its counterpart,GFP1-10, through an available Gal4 driver line. The unique aspect of this system is that neither GFP1-10nor GFP11alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP11tagged endogenous protein. We illustrate this technique using the adhesion molecule geneteneurin-m(Ten-m) as a model, highlighting the generation and validation of GFP11protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

Cell-type-specific labeling of endogenous proteins using the split GFP system in Drosophila.

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.

Experience alters brain structure, but the underlying mechanism remained unknown. Structural plasticity reveals that brain function is encoded in generative changes to cells that compete with destructive processes driving neurodegeneration. At an adult critical period, experience increases fiber number and brain size in Drosophila. Here, we asked if Toll receptors are involved. Tolls demarcate a map of brain anatomical domains. Focusing on Toll-2, loss of function caused apoptosis, neurite atrophy and impaired behaviour. Toll-2 gain of function and neuronal activity at the critical period increased cell number. Toll-2 induced cycling of adult progenitor cells via a novel pathway, that antagonized MyD88-dependent quiescence, and engaged Weckle and Yorkie downstream. Constant knock-down of multiple Tolls synergistically reduced brain size. Conditional over-expression of Toll-2 and wek at the adult critical period increased brain size. Through their topographic distribution, Toll receptors regulate neuronal number and brain size, modulating structural plasticity in the adult brain.

The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance may be evolutionarily conserved within insects.

Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled.

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.

The ability of cells to receive and process chemical information is fundamental to the function of biological systems. Cell responses to salient chemical cues are characteristically reliable and precise, a striking example being the guidance of axons to their targets during brain development. However, chemical signalling pathways are subject to multiple sources of unavoidable noise, due to the random nature of molecular motion and interactions. This suggests that cellular systems may be optimised for transduction of noisy signals, which provides a guiding principle for understanding cell function at a biophysical level. In pursuit of this idea, we study three model problems, motivated by sensing and signalling in axon guidance. Our approach is to construct mathematical models, and through analysis and simulation, extract general principles and hypotheses about the quantitative nature of biological noise and mechanisms for sensitive chemosensation.First, we quantify the physical limits of chemosensation imposed by the random thermal motion of the molecules that need to be counted. In particular, we show how this depends on the dimension and spatial extent of the domain of diffusion. Although recurrent diffusion in 1d and 2d is detrimental to measurement precision, we find these effects are suppressed when sensing is performed within a confined space. Second, we study the stochastic behaviour of the inositol 1,4,5-trisphosphate receptor ion channel, which couples receptor activity at the membrane to the downstream calcium response that regulates axon growth and turning. By modelling the basic biophysical events that control ion channel opening, we introduce a new principle for understanding the origin of the multiple gating modes observed in single channel recordings. Third, we examine the finely-tuned response of dorsal root ganglia neurons to very shallow neurotrophin gradients. We show how paracrine signalling within the ganglion could explain this extreme sensitivity, as well as the currently unexplained biphasic dose response of many axon growth and guidance cues.Overall, this thesis gives new quantitative insights into chemical signalling in biological systems, across multiple stages of processing and spatial scales. Our models make testable predictions to motivate new experiments, and provide a strong foundation for further theoretical and computational study in both axon guidance and broader domains of biophysics.

As neurons begin to differentiate, they send out processes called axons to initiate the formation of functional nerve connections. A specialized structure at the end of an axon called the growth cone is believed to possess the impressive navigational and target recognition ability crucial for this process. The goal of the research presented in this thesis was to understand the cellular and molecular mechanisms that shape axon growth and guidance in vivo during early brain development using a multifaceted experimental approach. Towards this goal we employed the simple, well-characterized neuronal scaffold of the embryonic zebrafish brain in combination with cell labeling techniques and performed studies in three specific areas: (1) the dynamic behaviors of navigating growth cones to obtain information about their cellular interactions with each other and the local environment, (2) the action of specific proteins (netrin and its receptor DCC) known to be involved in axon guidance in order to determine their function in vivo, and (3) the mobility of GFP in growth cones as a way to gain insight into the dynamics of molecular species in these structures as they actively navigate. Critical to our studies was a stable transgenic gata2::GFP zebrafish line in which we found high level of GFP expression in early forebrain neuronal clusters allowing in vivo timelapse study of the growth cones that pioneer the postoptic commissural (POC) axon tract. Following the development of the POC also allowed us to investigate how early commissural growth cones behave at the midline. Timelapse analysis of POC axon kinetics revealed important insight into growth cone interactions with each other and their environment and showed that these have behavioral consequences. While it was known that commissural axons slow down while crossing the midline, our data showed that this is only true for the leader axons. Follower axons do not slow down unless the leader axon is ablated. Together this analysis revealed that in addition to specific molecular cues, axon-axon interactions are important for establishing early axon tract. This characterization of POC axon kinetics and growth cone behavior in turn, provided us with an assay for studying the specific role of netrin, primarily a midline attractant for commissural axons in the spinal cord and its receptor, deleted in colorectal cancer (DCC). Loss- and gain-of-function experiments in combination with timelapse imaging uncovered a novel function for netrin as a positional repellent cue for POC axons. Finally, prompted by the observed differences in leader and follower POC growth cones, we developed a new experimental approach to assay GFP mobility as a reporter for the diffusion rates of other molecular species inside growth cones in vivo. We found that diffusion rates in actively pioneering growth cones are significantly decreased compared to follower axons suggesting that diffusion rates might be linked to growth cone pathfinding. Collectively, the findings presented in this thesis constitute a framework that allows for an integrative approach of studying growth cone navigation in vivo. Basic integrative knowledge of this sort is expected to aid the development of medical therapies related to nerve injury and repair.

The plasma membrane of cells contains a variety of lipid and protein molecules that are often segregated and heterogeneously distributed in microdomains. Lipid rafts represent a generalized concept of membrane microdomains that are enriched in cholesterol and sphingolipids and, characteristically, resistant to cold detergent extraction. Lipid rafts have recently received considerable attention because they are thought to be involved in many cellular functions, in particular, signal transduction for extracellular stimuli. Many of these functions are also intimately related to the processes involved in neural development, including neurotrophic factor signaling and synaptic plasticity. Recent studies from our lab and others have indicated an important role for lipid rafts in axonal growth and guidance. Specifically, our data show that lipid rafts on the plasma membrane provide platforms for spatial and temporal control of guidance signaling by extracellular cues. In addition, lipid rafts may also function in other aspects of axonal growth and guidance, including spatial and temporal regulation of adhesion, cytoskeletal dynamics, and growth cone motility. Further elucidating how membrane rafts are involved in guided axonal growth would provide important insights into the intricate signaling mechanisms underlying neuronal wiring, which is fundamental for normal brain development and functional recovery after injury and diseases.

Glia and neurons are intimately associated throughout bilaterian nervous systems, and were early proposed to interact for patterning circuit assembly. The investigations of circuit formation progressed from early hypotheses of intermediate guideposts and a “glia blueprint”, to recent genetic and cell manipulations, and visualizations in vivo. An array of molecular factors are implicated in axon pathfinding but their number appears small relatively to circuit complexity. Comprehending this circuit complexity requires to identify unknown factors and dissect molecular topographies. Glia contribute to both aspects and certain studies provide molecular and functional insights into these contributions. Here, I survey glial roles in guiding axon navigation in vivo, emphasizing analogies, differences and open questions across major genetic models. I highlight studies pioneering the topic, and dissect recent findings that further advance our current molecular understanding. Circuits of the vertebrate forebrain, visual system and neural tube in zebrafish, mouse and chick, the Drosophila ventral cord and the C. elegans brain-like neuropil emerge as major contexts to study glial cell functions in axon navigation. I present astroglial cell types in these models, and their molecular and cellular interactions that drive axon guidance. I underline shared principles across models, conceptual or technical complications, and open questions that await investigation. Glia of the radial-astrocyte lineage, emerge as regulators of axon pathfinding, often employing common molecular factors across models. Yet this survey also highlights different involvements of glia in embryonic navigation or pioneer axon pathfinding, and unknowns in the molecular underpinnings of glial cell functions. Future cellular and molecular investigations should complete the comprehensive view of glial roles in circuit assembly.

Nervous system development