Cracking the combinatorial semaphorin code

In this issue of Neuron, Wu et?al. describe a combinatorial code of repulsive Sema-2a and attractive Sema-2b signaling that mediates mechanosensory axonal guidance, fasciculation, and synaptic target selection within the CNS of Drosophila. Their work exemplifies how a detailed, multilevel molecular-genetic analysis (from molecules to behavior) provides fundamental insights into neural circuit development.     

A combinatorial code for the interaction of alpha-synuclein with membranes

Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.     

Persistence of Hunchback in the terminal region of the Drosophila blastoderm embryo impairs anterior development

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterised Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity.     

Motor pattern selection by combinatorial code of interneuronal pathways

We use a modeling approach to examine ideas derived from physiological network analyses, pertaining to the switch of a motor control network between two opposite control modes. We studied the femur–tibia joint control system of the insect leg, and its switch between resistance reflex in posture control and “active reaction” in walking, both elicited by the same sensory input. The femur–tibia network was modeled by fitting the responses of model neurons to those obtained in animals. The strengths of 16 interneuronal pathways that integrate sensory input were then assigned three different values and varied independently, generating a database of more than 43 million network variants. We demonstrate that the same neural network can produce the two different behaviors, depending on the combinatorial code of interneuronal pathways. That is, a switch between behaviors, such as standing to walking, can be brought about by altering the strengths of selected sensory integration pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heterotopic transplantation in the syncytial blastoderm ofDrosophila: Evidence for anterior and posterior nuclear commitments

Summary The interaction ofDrosophila syncytial blastoderm nuclei and cortical cytoplasm in the control of somatic developmental commitments was studied by transplanting genetically marked nuclei and surrounding cytoplasm between anterior and posterior flanks. After completion of cellularization the host egg was cut. Host anterior or posterior partial embryos were cultured in adult abdomens for 8–10 days, then the larval tissue removed and injected into larval hosts for metamorphosis. Differentiated ectodermal implants were recovered from emerged adults and characterized. One hundred sixteen clearly interpretable control and experimental implants were found. Of the 73 experimental implants 15 were derived from donor nuclei.Among the 15 donor implants, 14 autonomously formed donor site anterior (head and thoracic) or posterior (abdomen and genital) structures. This donor autonomy is interpreted to mean that nuclear and cytoplasmic factors necessary for anterior and posterior somatic commitments are present and transplantable prior to the completion of cellularization. Since donor nuclei injected directly into host flanks, or premixed with host cytoplasm, would have been well exposed to any host cytoplasmic factors, donor nuclei appear to have adopted anterior or posterior somatic commitments which are stable to significant cytoplasmic alterations.In 14 implants, host nuclei exposed to donor material altered somatic fate and formed donor type structures. These conversions are interpreted to imply that cytoplasmic factors controlling anterior or posterior somatic fates are present in the syncytial balstoderm embryo.     

Spatial regulation of segment polarity gene expression in the anterior terminal region of the Drosophila blastoderm embryo

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene.     

Calcium-independent adhesion of extra-embryonic endoderm cells from the early chick blastoderm is inhibited by the blastoderm beta-D-galactoside-binding lectin and by beta-galactosidase

Extra-embryonic endoderm cells from gastrulating chick embryos possess Ca2+-dependent and Ca2+-independent adhesive mechanisms. These cells also contain an endogenous beta-D-galactoside-binding lectin and cell surface receptors bearing galactose groups. The endogenous lectin inhibits cellular adhesion. To test whether the adhesive interactions involving lectin and galactose molecules are part of the Ca2+-independent or Ca2+-dependent adhesive mechanism, dissociated cells which were preincubated in beta-galactosidase were allowed to aggregate in the presence and absence of Ca2+ ions. Significant decreases in adhesion were observed in both cases. Cells were also allowed to aggregate in the presence and absence of Ca2+ ions when blastoderm lectin was present in the medium. Adhesion was decreased in both cases. The results suggest that cell surface galactose groups and the beta-D-galactoside-binding lectin are involved in Ca2+-independent adhesion.     

A combinatorial semaphorin code instructs the initial steps of sensory circuit assembly in the Drosophila CNS

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.