Developmental expression of the lipocalin Lazarillo and its role in axonal pathfinding in the grasshopper embryo.

This article describes the expression pattern and functional analysis of Lazarillo, a novel cell surface glycoprotein expressed in the embryonic grasshopper nervous system, and a member of the lipocalin family. Lazarillo is expressed by a subset of neuroblasts, ganglion mother cells and neurons of the central nervous system, by all sensory neurons of the peripheral nervous system, and by a subset of neurons of the enteric nervous system. It is also present in a few non neuronal cells associated mainly with the excretory system. A monoclonal antibody raised against Lazarillo perturbs the extent and direction of growth of identified commissural pioneer neurons. We propose that Lazarillo is the receptor for a midline morphogen involved in the outgrowth and guidance of these neurons.

A Resource for Manipulating Gene Expression and Analyzing cis-Regulatory Modules in the Drosophila CNS

Stem Cell Factor Functions as an Outgrowth-Promoting Factor to Enable Axon Exit from the Midline Intermediate Target

Neuronal determination during embryonic development of the grasshopper nervous system

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.

The generation of cellular diversity in the developing embryonic central nervous system of Drosophila melanogaster requires the precise orchestration of several convergent molecular and cellular mechanisms. Most reviews have focused on the formation and specification of neuroblasts (NBs), the putative neural stem cell in the Drosophila central nervous system. NBs divide asymmetrically to regenerate themselves and produce a secondary precursor cell called a ganglion mother cell (GMC), which divides to produce neurons and glia. Historically, our understanding of GMC specification has arisen from work involving asymmetric localization of intrinsic factors in the NB and GMC. However, recent information on NB lineages has revealed additional intrinsic factors that specify general and specific GMC fates. This review addresses what has been revealed about these intrinsic cues with regard to GMC specification. For example, Prospero, an asymmetrically localized determinant, plays a general role to enable GMC development and to distinguish GMCs from NBs. In contrast, the temporal gene cascade functions within NB lineages to ensure that each GMC in a lineage acquires a different fate. Two different mechanisms used to make the progeny of GMCs different will also be discussed. One is a generic mechanism, regulated by Notch and Numb, that allows sibling cells to adopt different fates. The other mechanism involves genes, such as even-skipped and klumpfuss that specify the fate of individual GMCs. All of these mechanisms converge within a GMC to bestow upon it a unique fate.

For an embryo to successfully develop into an adult animal, specific genes must act in different types of cells. Though all the cells have the same genes encoded within their DNA, looking at the way that the DNA is packaged can indicate which parts of the DNA are important for that particular cell type. If regions of DNA are “open” one can infer that those regions are actively involved in gene regulation, whereas “closed” regions are considered less important. It is currently difficult to determine which parts of the DNA are open within an individual cell type in a complex organ, such as the brain. Existing methods require the cells to be physically isolated from the tissue, which is technically challenging. To overcome this issue, Aughey et al. have now developed a method that does not require isolation of the cells. The new technique involves using genetic engineering to introduce an enzyme called Dam into specific cell types in living fruit flies. This enzyme adds a chemical label on regions of open DNA, which can then be detected. Aughey et al. tested this technique on various cells of the developing brain and gut, and were able to see differences in the openness of DNA that corresponded to the action of genes that are important in each cell type. The data also contain trends that help to understand the role of open DNA in development. For example, mature cells were shown to overall have less open DNA than the stem cells that divide to generate them. Aughey et al. hope their new technique will be of use to other researchers working with either fruit flies or mammalian tissues. The knowledge that scientists will gain from identifying how open DNA contributes to gene regulation, in both healthy and diseased tissues, will further our understanding of human development and the biology of diseases such as cancer.

The approximately 300 distinct neurons comprising each hemineuromere of the Drosophila embryonic central nervous system are derived from a segmentally reiterated array of approximately 30 progenitor cells, neuroblasts (NBs). Each NB has a unique identity and undergoes repeated cell divisions to produce several smaller secondary precursor cells, ganglion mother cells (GMCs); each GMC divides once to produce two neurons and/or glia, thereby generating a specific lineage of neurons/glia. Understanding the generation of neuronal diversity requires not only elucidation of the molecules and mechanisms that specify NB identity but also those that act to differentiate between the cell types produced within one NB lineage. Here we show that the Drosophila Zn finger protein Klumpfuss (Klu), which shows sequence similarities to the mammalian Wilm's tumor suppressor (WT-1), acts to differentiate between the identities of the first two secondary precursor cells produced from one NB lineage. Klu is expressed in the NB4-2 lineage only after two rounds of NB cell division, in the second born GMC (GMC4-2b). In loss-of-function mutant embryos, the first born GMC (GMC4-2a) as well as its progeny neurons are duplicated; we show that this duplication of the GMC4-2a sublineage arises because GMC4-2b adopts the identity of GMC4-2a and divides to produce the GMC4-2a progeny. Moreover, when Klu is ectopically expressed in GMC4-2a, it fails to acquire its normal identity and fails to produce correctly specified progeny. klu therefore acts to specify the identity of GMC4-2b and to make it distinct from GMC4-2a. Our findings further suggest that the determination of GMC cell fate occurs in two steps; the initial GMC identity is the consequence of inheritance from the maternal NB, however, the subsequent stabilization of this identity requires functions like klu in the GMC.

Chondroitin sulfate proteoglycans (CSPGs) play important roles in the developing and mature nervous system, where they guide axons, maintain stable connections, restrict synaptic plasticity, and prevent axon regeneration following CNS injury. The chondroitin sulfate glycosaminoglycan (CS GAG) chains that decorate CSPGs are essential for their functions. Through these sugar chains, CSPGs are able to bind and regulate the activity of a diverse range of proteins and through these interactions can regulate neuronal growth. These CS-protein interactions depend on specific sulfation patterns within the CS GAG chains, and accordingly, particular CS sulfation motifs are upregulated during development, in the mature nervous system, and in response to CNS injury. Thus, spatiotemporal regulation of CS GAG biosynthesis may provide an important mechanism to control the functions of CSPGs and modulate intracellular signaling pathways. Here, we will discuss these sulfation-dependent processes and highlight how the CS sugars on CSPGs contribute to neuronal growth, axon guidance, and plasticity in the nervous system. Chondroitin sulfate proteoglycans (CSPGs) are a major barrier to regenerating axons in the central nervous system (CNS), exerting their inhibitory effect through their polysaccharide side chains. Chondroitin sulfate (CS) potently inhibits axon regeneration through modulation of inhibitory signaling pathways induced by carbohydrate binding to protein ligands and receptors. Here, we identify a novel carbohydrate-protein interaction between CS and EphA4 that inhibits axon regrowth. We characterize the mechanism of activation and demonstrate how carbohydrate binding induces phosphorylation of the intracellular kinase domain through clustering of cell surface EphA4. Collectively, our studies present a novel mechanism of EphA4 activation by CS independent of the canonical ephrin ligands and uncover the role of this interaction in inhibition of neurite regrowth after injury. Our results underscore a mechanism of action by which carbohydrates can function as direct, activating ligands for protein receptors and provide mechanistic insights into the inhibition of axon growth by CS following injury to the CNS. Chondroitin sulfate proteoglycans (CSPGs) regulate neuronal plasticity, as well as axon regeneration and guidance through their ability to bind protein ligands and cell surface receptors. In this way, extracellular CSPGs can modulate the activity of intracellular signaling pathways. Here, a computational analysis of EphA4-CS interactions is performed to characterize the importance of key arginine and lysine residues towards CS binding, and to identify structural differences in CS-A, CS-C, CS-D, and CS-E docking to EphA4. Carbohydrate-induced Eph receptor clustering could be a general mechanism of Eph receptor activation. To identify additional Eph receptors that interact with CS, CS-E was docked to all EphA and EphB family members to predict relative binding affinities. The relative strengths of the predicted binding energies are: EphB4 > EphA8 > EphA1 > EphA3 > EphB1 > EphB3 > EphA7 > EphA5 > EphA4 > EphA6 > EphB2 > EphB6 > EphA2. In addition, the arginine and lysine residues that mediate CS binding are identified for each Eph receptor. These computational predictions provide mechanistic insights into Eph receptor activation by chondroitin sulfate and have implications for inhibition of axon regeneration following injury to the nervous system and axon guidance during development.

Tyrosine kinase signaling pathways are essential for process outgrowth and guidance during nervous system development. We have examined the roles of tyrosine kinase activity in programming growth cone guidance decisions in an intact nervous system in which neurons can be individually identified. We applied the tyrosine kinase inhibitors herbimycin A and genistein to whole 40% grasshopper embryos placed in medium, or injected the inhibitors into intact grasshopper eggs. Both inhibitors caused interneuronal axons that normally would grow along the longitudinal connectives to instead leave the central nervous system (CNS) within the segmental nerve root and grow out toward the body wall muscles. In addition, herbimycin A produced pathfinding errors in which many longitudinal axons crossed the CNS midline. To study how this drug affected guidance decisions made by individual growth cones, we dye-filled the pCC interneuron, which normally extends an axon anteriorly along the ipsilateral longitudinal connective. In the presence of herbimycin A, the pCC growth cone was redirected across the anterior commissure. These phenotypes suggest that tyrosine kinase inhibition blocks a signaling mechanism that repels the growth cones of longitudinal connective neurons and prevents them from crossing the midline.

Delaminated neuroblasts in Drosophila function as stem cells during embryonic central nervous system development. They go through repeated asymmetric divisions to generate multiple ganglion mother cells, which divide only once more to produce postmitotic neurons. Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. We show that the Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string.

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.

Brain neuroblasts in the embryonic grasshopper were studied by toluidine blue staining, BrdU incorporation, and immunocytochemistry in whole-mounts as well as by reconstruction of stained serial sections. Large dividing neuroblasts are observed by the 25% stage. During early neurogenesis these neuroblasts generate their progeny through mechanisms similar to those that occur in the segmental ganglia; each neuroblast divides asymmetrically to produce a chain of ganglion mother cells, and each ganglion mother cell divides symmetrically to produce a pair of neurons. Approximately 130 mitotically active, large neuroblasts are found in each brain hemisphere at the 30–45% stages. Through morphogenetic movements that occur between the 30–35% stages these neuroblasts become located in positions which are predictive of the major brain regions that they give rise to. Many of the brain neuroblasts can be identified as individuals based on their stereotyped position in the neurogenic array. Immunocytochemical experiments with antibodies against, engrailed, fasciclin I and TERM-1 show that brain neuroblasts can also be characterized by their expression of cell-specific molecular labels. These studies indicate that many features of the complex mature insect brain derive from a surprisingly simple and stereotyped set of neuronal precursor cells. Thus, many of the concepts and methods that have been used to study neurogenesis in the simpler segmental ganglia may also be applicable to the insect brain.

The embryonic lethal abnormal visual system (elav) gene of Drosophila melanogaster is required for the development and maintenance of the nervous system. Transcripts from this locus are distributed ubiquitously throughout the nervous system at all developmental stages. A product of this gene, the ELAV protein, has homology to known RNA binding proteins. The localization of the ELAV protein was studied in all developmental stages using antibodies that were generated against a hybrid protein made in Escherichia coli. In general, these data are consistent with previous results and demonstrate that (1) the ELAV protein is detected in the developing embryonic nervous system at a time coincident with the birth of the first neurons, (2) the ELAV protein is first detected in the majority of neurons of the central and peripheral nervous systems of embryos, larvae, pupae, and adults, (3) the ELAV protein appears to be localized to the nucleus, and (4) the ELAV protein is not detected in neuroblasts or identifiable glia. These data also provide new information concerning elav expression and show that (1) ELAV is not expressed in the ganglion mother cells (GMCs), (2) while the ELAV protein is localized to the nucleus, it is not uniformly distributed throughout this structure, and (3) other Drosophila species do express an ELAV-like antigen. We propose that the elav gene provides a neuronal-housekeeping function that is required for the successful posttranscriptional processing of transcripts from a set of genes the function of which is required for proper neuronal development and maintenance.

In the developing vertebrate brain neurons differentiate in largely invariant locations and axons project with remarkable precision to innervate specific targets. While the general principles of axon growth and guidance are understood in vitro, much less is known about these processes in vivo. Recent studies suggest that a complex interplay of chemorepulsive and chemoattractive cues are necessary for establishing the early scaffold of axon tracts in the vertebrate brain. This thesis reports the identification, characterisation and functional analysis of three putative axon guidance molecules: Xenopus Roundabout-1, zebrafish Roundabout-3 and brother of CDO (Cell adhesion molecule-related/Down regulated by Oncogenesis). All are members of the immunoglobulin (Ig) superfamily members and all are involved in the growth and guidance of axons in the embryonic vertebrate brain. The receptor Roundabout-1 (Robol) and its ligand Slit are known to infiuence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. This thesis reports the identification and expression of a Xenopus Robol1 orthologue that is highly homologous to mammalian Robo1. The role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain was investigated using overexpression studies, immunohistochemical and in situ hybridisation techniques. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robol in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain. In zebrafish, two forms of the putative axon guidance molecule roundabout-3 (Robo3) have been reported and are described in this thesis as Robo3a and 3b. The role of Robo3b in axon guidance in the zebrafish forebrain was analysed using immunohistochemical techniques, misexpression and antisense morpholino oligonucleotides. Robo3b is expressed in the main nuclei of the fore-, mid- and hindbrain as well as in cells outside of the nervous system. Analysis of Robo3b gain-of-function and loss-of-function mutant zebrafish embryos revealed defects in both dorsoventral and rostro-caudal projecting axon tracts. These results indicate that Robo3b is functionally active and plays select roles in axon navigation during formation of the early axon scaffold in the embryonic vertebrate brain. Brother of CDO has previously been shown to positively regulate myogenic differentiation in vitro and is expressed in a restricted pattern in the dorsal neural tube in the mouse. This thesis reports the isolation of a zebrafish orthologue of BOC (zboc) and describes its expression pattern, zboc is expressed in a variety of tissues firom early in development and its distribution is consistent with multiple functional roles. In the brain zboc is prominent in the ventricular margin of the dorsal neural tube. The role of zboc was first assessed in gain-of-function mutants following injection of zboc mRNA into one-cell embryos. These animals suffered severe defects in the patterning of the rostrocaudal axis typical of dorsalised embryos. Loss-of-function mutants were also produced using a zboc antisense morpholino oligonucleotide. Analysis of embryos injected at the one-cell stage revealed consistent defects in the dorsoventrally projecting axon tracts in the rostral brain. This thesis presents the first functional evidence for the role of Xenopus Roundabout-1, zebrafish Roundabout-3 and brother of CDO in the development of a stereotypical axon scaffold in the vertebrate brain. It provides a foundation for fiiture research into the complex interactions that control the growth and guidance of axons in vivo.n