Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis.

In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system.

A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion.

The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509-514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173-180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103-110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting.

Fasciclin I is a membrane-associated glycoprotein that is regionally expressed on a subset of fasciculating axons during neuronal development in insects; it is expressed on apposing cell surfaces, suggesting a role in specific cell adhesion. In this paper we show that Drosophila fasciclin I is a novel homophilic cell adhesion molecule. When the nonadhesive Drosophila S2 cells are transfected with the fasciclin I cDNA, they form aggregates that are blocked by antisera against fasciclin I. When cells expressing fasciclin I are mixed with cells expressing fasciclin III, another Drosophila homophilic adhesion molecule, the mixture sorts into aggregates homogeneous for either fasciclin I- or fasciclin III-expressing cells. The ability of these two novel adhesion molecules to mediate cell sorting in vitro suggests that they might play a similar role during neuronal development.

Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex.

The events of myoblast fusion in Drosophila are dissected here by combining genetic analysis with light and electron microscopy. We describe a new and essential intermediate step in the process, the formation of a prefusion complex consisting of "paired vesicles." These pairs of vesicles from different cells align with each other across apposed plasma membranes. This prefusion complex resolves into dense membrane plaques between apposed cells; these cells then establish cytoplasmic continuity by fusion of small areas of plasma membrane followed by vesiculation of apposed membranes. Different steps in this process are specifically blocked by mutations in four genes required for myoblast fusion. One of these genes, blown fuse, encodes a novel cytoplasmic protein expressed in unfused myoblasts that is essential for progression beyond the prefusion complex stage.

Neuron duplications and deletions in locust clones and clutches.

Duplications and deletions of identified neurons can occur with a high degree of genetic control and specificity, as shown by examining the ocellar interneurons of locusts from different breeding populations, clutches from single mated pairs, and isogenic animals from parthenogenetic clones.

Guidance of peripheral pioneer neurons in the grasshopper: adhesive hierarchy of epithelial and neuronal surfaces.

An important question in developmental neurobiology is how a neuron finds its way over long distances to its correct target during embryogenesis. Peripheral pioneer neurons in insect embryos have been used for study because of the relative simplicity of the early embryonic appendages, and the accessibility of the identified neurons whose growth cones traverse this terrain. The data presented suggest an adhesive hierarchy of both epithelial and neuronal surfaces that guides the first growth cones from the appendages of the grasshopper embryo.

The molecular biology of axon guidance.

Neuronal growth cones navigate over long distances along specific pathways to find their correct targets. The mechanisms and molecules that direct this pathfinding are the topics of this review. Growth cones appear to be guided by at least four different mechanisms: contact attraction, chemoattraction, contact repulsion, and chemorepulsion. Evidence is accumulating that these mechanisms act simultaneously and in a coordinated manner to direct pathfinding and that they are mediated by mechanistically and evolutionarily conserved ligand-receptor systems.

Growth cone guidance in insects: fasciclin II is a member of the immunoglobulin superfamily.

The cellular cues that guide neuronal growth cones toward their targets are highly conserved in such diverse organisms as insects and vertebrates. Evidence presented here suggests that the molecular mechanisms underlying these events may be equally conserved. This article describes the structure and function of fasciclin II, a glycoprotein expressed on a subset of fasciculating axons in the grasshopper embryo. Antibody perturbation experiments suggest that fasciclin II functions in mediating one form of neuronal recognition: selective fasciculation. Fasciclin II is a member of the immunoglobulin gene superfamily and is homologous in structure and function to the neural cell adhesion molecule N-CAM and to several other vertebrate cell adhesion molecules.

A neural tetraspanin, encoded by late bloomer, that facilitates synapse formation.

Upon contacting its postsynaptic target, a neuronal growth cone transforms into a presynaptic terminal. A membrane component on the growth cone that facilitates synapse formation was identified by means of a complementary DNA-based screen followed by genetic analysis. The late bloomer (lbl) gene in Drosophila encodes a member of the tetraspanin family of cell surface proteins. LBL protein is transiently expressed on motor axons, growth cones, and terminal arbors. In lbl mutant embryos, the growth cone of the RP3 motoneuron contacts its target muscles, but synapse formation is delayed and neighboring motoneurons display an increase in ectopic sprouting.

Switching repulsion to attraction: changing responses to slit during transition in mesoderm migration.

Slit is secreted by cells at the midline of the central nervous system, where it binds to Roundabout (Robo) receptors and functions as a potent repellent. We found that migrating mesodermal cells in vivo respond to Slit as both an attractant and a repellent and that Robo receptors are required for both functions. Mesoderm cells expressing Robo receptors initially migrate away from Slit at the midline. A few hours after migration, these same cells change their behavior and require Robo to extend toward Slit-expressing muscle attachment sites. Thus, Slit functions as a chemoattractant to provide specificity for muscle patterning.

Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis.

Segmentation genes control cell identities during early pattern formation in Drosophila. One of these genes, fushi tarazu (ftz), is now shown also to control cell fate during neurogenesis. Early in development, ftz is expressed in a striped pattern at the blastoderm stage. Later, it is transiently expressed in a specific subset of neuronal precursor cells, neurons (such as aCC, pCC, RP1, and RP2), and glia in the developing central nervous system (CNS). The function of ftz in the CNS was determined by creating ftz mutant embryos that express ftz in the blastoderm stripes but not in the CNS. In the absence of ftz CNS expression, some neurons appear normal (for example, the aCC, pCC, and RP1), whereas the RP2 neuron extends its growth cone along an abnormal pathway, mimicking its sibling (RP1), suggesting a transformation in neuronal identity.

Embryonic development of identified neurons: temporal pattern of morphological and biochemical differentiation.

Individually identified neurons can be recognized in grasshopper embryos, and are accessible to examination by morphological, physiological, and biochemical techniques from their birth to their maturation. Only after the axon of an identified neuron reaches its postsynaptic target does the neurotransmitter accumulate, the soma rapidly enlarge, and the central arborizations greatly expand.

Cell recognition during neuronal development.

Insect embryos, with their relatively simple nervous systems, provide a model system with which to study the cellular and molecular mechanisms underlying cell recognition during neuronal development. Such an approach can take advantage of the accessible cells of the grasshopper embryo and the accessible genes of Drosophila. The growth cones of identified neurons express selective affinities for specific axonal surfaces; such specificities give rise to the stereotyped patterns of selective fasciculation common to both species. These and other results suggest that early in development cell lineage and cell interactions lead to the differential expression of cell recognition molecules on the surfaces of small subsets of embryonic neurons whose axons selectively fasciculate with one another. Monoclonal antibodies reveal surface molecules in the Drosophila embryo whose expression correlates with this prediction. It should now be possible to isolate the genes encoding these potential cell recognition molecules and to test their function through the use of molecular genetic approaches in Drosophila.

Targeted disruption of Ca(2+)-calmodulin signaling in Drosophila growth cones leads to stalls in axon extension and errors in axon guidance.

Ca(2+)-calmodulin (CaM) function was selectively disrupted in a specific subset of growth cones in transgenic Drosophila embryos in which a specific enhancer element drives the expression of the kinesin motor domain fused to a CaM antagonist peptide (kinesin-antagonist or KA, which blocks CaM binding to target proteins) or CaM itself (kinesin-CaM or KC, which acts as a Ca(2+)-binding protein). In both KA and KC mutant embryos, specific growth cones exhibit dosage-dependent stalls in axon extension and errors in axon guidance, including both defects in fasciculation and abnormal crossings of the midline. These results demonstrate an in vivo function for Ca(2+)-CaM signaling in growth cone extension and guidance and suggest that Ca(2+)-CaM may in part regulate specific growth cone decisions, including when to defasciculate and whether or not to cross the midline.

Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance.

We used the enhancer detection/GAL4 system in Drosophila to direct increased levels of Fasciclin II (Fas II) expression on motoneuron growth cones and axons and to direct ectopic Fas II expression on other cells they encounter. Four classes of abnormal phenotypes are observed: "bypass" phenotypes, in which axons fail to defasciculate at the choice point where they would normally enter their muscle target region and instead extend past their target; "detour" phenotypes, in which these bypass growth cones enter their muscle target region at a different location; "stall" phenotypes, in which axons that enter their muscle target region fail to defasciculate from one another to probe their muscle targets; and "misroute" phenotypes, in which growth cones are diverted onto abnormal pathways by contact with Fas II-positive cells. These phenotypes show that changes in the pattern and level of Fas II expression can alter growth cone guidance, apparently in part by modulating the ability of these growth cones to respond to other guidance cues.

Ectopic expression of connectin reveals a repulsive function during growth cone guidance and synapse formation.

Connectin, a cell surface protein of the leucine-rich repeat family in Drosophila, is expressed on the surface of a subset of embryonic muscles (primarily lateral muscles), on the growth cones and axons of the motoneurons that innervate these muscles (primarily SNa motoneurons), and on several associated glial cells. When coupled with its ability to mediate homophilic cell adhesion, these results led to the suggestion that Connectin functions as an attractive signal for SNa pathfinding and targeting. In the present study, we ectopically expressed Connectin on ventral muscles normally innervated by SNb motoneurons. The SNb growth cones change both their morphology and their trajectory when they encounter ectopic Connectin-positive ventral muscles, displaying "bypass," "detour," and "stall" phenotypes. Moreover, SNb synapse formation is prevented by Connectin expression on ventral muscles. These results reveal a repulsive function for Connectin during motoneuron growth cone guidance and synapse formation.

Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.

We developed a rapid method that uses diphtheria toxin, the flp recognition target sequences, and the GAL4-UAS activation system, to ablate specific neurons in the Drosophila embryo and to examine the consequences in large numbers of embryos at many time points. We used this method to show that, in the absence of the aCC axon, which pioneers the intersegmental nerve in the PNS, the three U follower axons are delayed and make frequent errors. However, the pathway ultimately forms in most segments. We also ablated the axons that pioneer the first longitudinal pathways within the CNS and observed similar results; the formation of longitudinal pathways is delayed and disorganized in 70% of segments, but these tracts ultimately form in 80% of segments. Thus, pioneers facilitate the development of PNS and CNS axon pathways; in their absence, followers are delayed and make numerous errors. However, pioneers are not absolutely required, as these embryos display a remarkable ability to correct for the loss of the pioneering neurons.

Genetic analysis of the mechanisms controlling target selection: target-derived Fasciclin II regulates the pattern of synapse formation.

In Drosophila, motoneuron growth cones initially probe many potential muscle targets but later withdraw most of these contacts to form stereotypic synapses with only one or a few muscles. Prior to synapse formation, Fasciclin II (Fas II) is expressed at low levels on muscle. During synapse formation, Fas II concentrates at the synapse and disappears from the rest of the muscle. We previously showed that Fas II is required both pre- and postsynaptically for synaptic stabilization. Here, we show that the differential expression of target-derived Fas II has a profound influence on the patterning of synapse formation. A transient increase in muscle Fas II stabilizes growth cone contacts and leads to novel synapses that are functional and stable; targets that normally receive two inputs can now receive up to six inputs. Changing the relative levels of Fas II on neighboring muscles leads to dramatic shifts in target selection.

Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling.

Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Previous studies showed that Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. Here, we define a seven amino acid sequence in PlexB required for Rac(GTP) binding. The interaction of PlexB with Rac(GTP) is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters Rac(GTP) from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to Rac(GTP) and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA.

Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity.

Increased cAMP (in dunce mutants) leads to an increase in the structure and function of the Drosophila neuromuscular junction. Synaptic Fasciclin II (Fas II) controls this structural plasticity, but does not alter synaptic function. Here, we show that CREB, the cAMP response element-binding protein, acts in parallel with Fas II to cause an increase in synaptic strength. Expression of the CREB repressor (dCREB2-b) in the dunce mutant blocks functional but not structural plasticity. Expression of the CREB activator (dCREB2-a) increases synaptic strength only in FasII mutants that increase bouton number. This CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release. Expression of dCREB2-a in a FasII mutant background genetically reconstitutes this cAMP-dependent plasticity. Thus, cAMP initiates parallel changes in CREB and Fas II to achieve long-term synaptic enhancement.