Christian Klämbt

The midline of the drosophila central nervous system: A model for the genetic analysis of cell fate, cell migration, and growth cone guidance

A row of mesectodermal cells separates the two lateral neurogenic regions in the Drosophila embryo and generates a discrete set of glia and neurons. Most CNS growth cones initially head straight toward the midline, suggesting that these midline cells play a key role in the formation of the axon commissures. We have used antibodies that stain the first axons, beta-galactosidase enhancer trap lines that selectively stain the different midline cells, and electron microscopic studies to elucidate the cells and interactions that mediate the normal formation of the two major commissures in each segment. This analysis has led to a model that proposes a series of sequential cell interactions controlling the development of the axon commissures. A genetic test of this model has utilized a number of mutations that, by either eliminating or altering the differentiation of various midline cells, perturb the development of the axon commissures in a predictable fashion.

Drosophila Futsch/22C10 Is a MAP1B-like Protein Required for Dendritic and Axonal Development

Here we report the description of the Drosophila gene futsch, which encodes a protein recognized by the monoclonal antibody 22C10 that has been widely used to visualize neuronal morphology and axonal projections. The Futsch protein is 5327 amino acids in length. It localizes to the microtubule compartment of the cell and associates with microtubules in vitro. The N- and C-terminal domains of Futsch are homologous to the vertebrate MAP1B microtubule-associated protein. The central domain of the Futsch protein is highly repetitive and shows sequence similarity to neurofilament proteins of which no Drosophila homologs have been reported. Loss-of-function analyses demonstrate that during embryogenesis Futsch is necessary for dendritic and axonal growth. Gain-of-function analyses demonstrate a functional interaction of Futsch with other MAPs. In addition, we show that during development, futsch expression is negatively regulated in nonneuronal tissues.

The argos gene encodes a diffusible factor that regulates cell fate decisions in the drosophila eye

The argos gene encodes a protein that is required for viability and that regulates the determination of cells in the Drosophila eye. A developmental analysis of argos mutant eyes indicates that the mystery cells, which are usually nonneuronal, are transformed into extra photoreceptors, and that supernumerary cone cells and pigment cells are also recruited. Clonal analysis indicates that argos acts nonautonomously and can diffuse over the range of several cell diameters. Conceptual translation of the argos gene suggests that it encodes a secreted protein.

The Ets transcription factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic CNS

The Drosophila gene pointed (pnt) encodes two putative transcription factors (P1 and P2) of the Ets family, which in the embryonic CNS are found exclusively in glial cells. Loss of pnt function leads to poorly differentiated glial cells and a marked decrease in the expression of the neuronal antigen 22C10 in the MP2 neurons, which are known to interact intimately with the pntP1-expressing longitudinal glial cells. Ectopic expression of pntP1 RNA forces additional CNS cells to enter the glial differentiation pathway. Interestingly, the additional glial-like cells are often flanked by cells that ectopically express the neuronal antigen 22C10. Therefore, both the pnt loss-of-function as well as the gain-of-function phenotype suggest that glial cells are able to induce 22C10 expression on neighboring neurons. This was further verified by cell transplantation experiments. Thus, pnt is not only required but also sufficient for several aspects of glial differentiation.

Organization and Function of the Blood–Brain Barrier in Drosophila

The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. Here, we present a detailed cellular analysis of the glial cell complement constituting the blood–brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, we characterize different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella. To test the functional roles of these sheaths we performed a series of dye penetration experiments in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants allowed to conclude that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). We further compare the requirements of different septate junction components for the integrity of the blood–brain barrier and provide evidence that two of the six Claudin-like proteins found in Drosophila are needed for normal blood–brain barrier function.

Epidermal growth factor receptor signaling.

Inhibitory signaling is an important way of fine tuning EGFR activity to enable a cell to discriminate between short and prolonged exposure to signaling molecules. Over the last few years a number of different mechanisms have been shown to abrogate receptor activity (Fig. 3Fig. 3). First, inhibitory signaling molecules are expressed in response to EGFR activation. The most prominent example is encoded by the Drosophila gene argos. Argos is a secreted molecule that has a single EGF-like domain with an expanded B-loop. Binding experiments showed that Argos binds directly to the EGFR presumably via its EGF-like domain and inhibits EGFR activity. Argos prevents Spitz binding and may also interfere with ligand-independent activation of the EGFR by preventing the formation of active EGFR dimers.Fig. 3Negative regulators of EGFR signaling. Several proteins were found to attenuate EGFR signaling. Argos, Decorin, and Kekkon bind to the extracellular domain of the EGFR. PKC is able to phosphorylate the EGFR at T654. Binding of Cbl at Y1045 results in ubiquitination and subsequent degradation. The recently identified Echinoid protein interferes with EGFR signaling downstream. LRR, leucine-rich repeat; Ig, immunoglobulin-like domain; FnIII, fibronectin type III repeat.View Large Image | View Hi-Res Image | Download PowerPoint SlideBesides Argos a number of other molecules attenuate EGFR activity. Again Drosophila has been a powerful system for identifying new components which can inhibit EGFR. Kekkon, which is expressed in response to EGFR activity, encodes a type I transmembrane protein and resembles cell adhesion molecules. The extracellular domain of Kekkon binds to the EGFR and provides a mechanism for negative-feedback. Two additional Kekkon-like proteins are encoded by the Drosophila genome which await characterisation.The Echinoid protein shows identity to the L1 adhesion protein and defines an additional pathway for antagonizing EGFR activity downstream of the receptor. EGFR activity can also be influenced by components of the extracellular matrix. One example is the leucine-rich proteoglycan Decorin that can bind EGFR and attenuate EGFR phosphorylation.sprouty and the cbl are genes which encode global inhibitors of EGFR signaling. Sprouty contains a conserved cysteine-rich domain and associates with components of the EGFR signaling cascade such as Grb2/Drk and GAP1. The function of Sprouty is, however, not restricted to the EGFR pathway. Cbl contains an unusual SH2 domain that is responsible for EGFR binding via Y1045. This interaction leads to the phosphorylation of Cbl, which may facilitate the ubiquitin ligase activity of the Cbl RING finger domain. Ubiquitination of the EGFR leads to its internalization via clathrin-coated vesicles and subsequent degradation.In summary, EGFR signaling appears to be integrated in a complex regulatory network linking extracellular signals to the cytoplasm and the nucleus. In the last decade the modulation of EGFR activity by negative regulators has become more and more evident. Now the picture is getting even more complicated by the fact that differential trafficking of activated EGFR may lead to different cellular responses. Further studies may not only help to increase our understanding of the role of EGFR signaling in the developing animal, they may ultimately lead to the development of efficient anti-cancer therapeutics.

Control of midline glia development in the embryonic Drosophila CNS.

The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord of Drosophila. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on the activation of the Drosophila EGF-receptor homolog and subsequent ras1/raf-mediated signal transduction. Nuclear targets of this signalling cascade are the ETS domain transcription factors pointedP2 and yan. In the midline glia pointedP2 in turn activates the transcription of argos, which encodes a diffusible negative regulator of EGF-receptor signalling.

Kette regulates actin dynamics and genetically interacts with Wave and Wasp

During development of the Drosophila nervous system, kette is required for axonal growth and pathfinding. It encodes a highly conserved homolog of the Nck-associated protein 1 (NAP1) that genetically interacts with the Drosophila homolog of Nck, dock. We show that in vivo as well as in tissue culture models most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. The kette mutant phenotype can be suppressed by reducing the wave gene dose, demonstrating that kette antagonizes wave function. Overexpression of the wild-type Kette protein does not interfere with normal development, whereas expression of an activated, membrane-tethered Kette protein induces the formation of large F-actin bundles in both, tissue culture cells and in vivo. This gain-of-function phenotype is independent of wave but can be suppressed by reducing the wasp gene dose, indicating that Kette is able to regulate Wasp, to which it is linked via the Abelson interactor (Abi). Our data suggest a model where Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization.

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila.

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.

The function of leak and kuzbanian during growth cone and cell migration

Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. Here we present an analysis of the genes leak and kuzbanian and show that both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, which encodes the Roundabout-2 Slit receptor, were first recovered by Nüsslein-Volhard and co-workers based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may be able to act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian also regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. Our analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex.