The midline glial cells are required for regionalization of commissural axons in the embryonic CNS of Drosophila

 The ventral nerve cord of arthropods is characterised by the organisation of major axon tracts in a ladder-like pattern. The individual neuromeres are connected by longitudinal connectives whereas the contra-lateral connections are brought about through segmental commissures. In each neuromere of the embryonic central nervous system (CNS) of Drosophila an anterior and a posterior commissure is found. The development of these commissures requires a set of neurone-glia interactions at the midline. Here we show that both the anterior as well as the posterior commissures are subdivided into three axon-containing regions. Electron microscopy of the ventral nerve cord of mutations affecting CNS midline cells indicates that the midline glial cells are required for this subdivision. In addition the midline glial cells appear required for a crossing of commissural growth cones perpendicular to the longitudinal tracts, since in mutants with defective midline glial cells commissural axons frequently cross the midline at aberrant angles. Received: 6 July 1997 / Accepted: 27 August 1997     

Commissure formation in the embryonic CNS of Drosophila.

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. In later stages, midline glial cells are required to separate anterior and posterior commissures into distinct axon bundles. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, we conducted a saturating ethylmethane sulfonate mutagenesis, screening for mutations which disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. In addition to commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene, two gene functions are required for the initial formation of commissural tracts. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures.     

Glia development in the embryonic CNS of Drosophila.

Glial cells are pivotal players during the development and function of complex nervous systems. In Drosophila, recent genetic analyses have revealed several genes that control differentiation and function of CNS glial cells and their interactions with neurons can be studied in detail at the CNS midline, where it is essential for the correct establishment of the commissural axon pattern.     

Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS

Whereas considerable progress has been made in understanding the molecular mechanisms of axon guidance across the midline, it is still unclear how the axonal trajectories of longitudinal pioneer neurons, which never cross the midline, are established. Here we show that longitudinal glia of the embryonic Drosophila CNS direct formation of pioneer axon pathways. By ablation and analysis of glial cells missing mutants, we demonstrate that glia are required for two kinds of processes. Firstly, glia are required for growth cone guidance, although this requirement is not absolute. We show that the route of extending growth cones is rich in neuronal cell bodies and glia, and also in long processes from both these cell types. Interactions between neurons, glia and their long processes orient extending growth cones. Secondly, glia direct the fasciculation and defasciculation of axons, which pattern the pioneer pathways. Together these events are essential for the selective fasciculation of follower axons along the longitudinal pathways.     

Inscuteable-independent apicobasally oriented asymmetric divisions in the Drosophila embryonic CNS

Inscuteable is the founding member of a protein complex localised to the apical cortex of Drosophila neural progenitors that controls their asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterised to date, in both the central and peripheral nervous systems, require inscuteable. Here we examine the generality of this requirement. We show that many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, we focused on the MP2 > dMP2 + vMP2 division. We show that for MP2 divisions, asymmetric localisation and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. We conclude that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable.     

Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS

On each side of the midline of the Drosophila CNS, axons are organized into a series of parallel pathways. Here we show that the midline repellent Slit, previously identified as a short-range signal that regulates midline crossing, also functions at long range to pattern these longitudinal pathways. In this long-range function, Slit signals through the receptors Robo2 and Robo3. Axons expressing neither, one, or both of these receptors project in one of three discrete lateral zones, each successively further from the midline. Loss of robo2 or robo3 function repositions axons closer to the midline, while gain of robo2 or robo3 function shifts axons further from the midline. Local cues further refine the lateral position. Together, these long- and short-range guidance cues allow growth cones to select with precision a specific longitudinal pathway.     

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.     

Specific expression of the Drosophila midline-jumper retro-transposon in embryonic CNS midline cells

Here we describe of a novel Drosophila LTR-type retrotransposon that is expressed in the embryonic CNS midline glia and in the embryonic germ cells. The element is related to the gypsy and burdock retrotransposons and was termed midline-jumper. In addition to cDNA clones generated from internal retrotransposon sequences, we have identified one cDNA clone that appears to reflect a transposition event, indicating that the midline-jumper retrotransposon is not only transcribed but also able to transpose during Drosophila development.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

Energy transfer from astrocytes to axons: the role of CNS glycogen

We tested the hypothesis that astrocytic glycogen supports axon function under both pathological and physiological conditions. Functional activity of the rat (RON) or mouse optic nerve (MON), representative central white matter tracts, was assessed electrophysiologically as the area under the supramaximal compound action potential (CAP). During aglycaemia the CAP area of rodent optic nerve persisted for up to 30 min, after which the CAP rapidly failed. Glycogen content measured biochemically during the aglycaemic insult fell with a time course compatible with its rapid degradation in the absence of glucose. Pharmacological up-regulation of glycogen content prior to the aglycaemic insult with incubation in hyperglycaemic ambient glucose delayed CAP failure, whereas down-regulation of glycogen content induced by nor-adrenaline accelerated CAP failure. Inhibiting lactate transfer between astrocytes and axons during aglycaemia, where glycogen is the only utilisable energy reserve, resulted in accelerated CAP failure, implying that glycogen-derived lactate supports function when exogenous energy metabolites are withdrawn. Under normoglycaemic conditions glycogen content decreased during high frequency axon discharge, although CAP function was fully maintained. Both prior depletion of glycogen content, or blocking axonal lactate uptake rendered nerves incapable of fully supporting CAP function during high frequency firing in the presence of normoglycaemic glucose. These results indicated that during aglycaemia and increased metabolic demand, astrocytic glycogen was degraded to form lactate, which was used as a supplemental energy source when ambient normoglycaemic glucose was incapable of meeting immediate tissue energy demands.     

Calmodulin and son of sevenless dependent signaling pathways regulate midline crossing of axons in the Drosophila CNS

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline.     

Courtship in Drosophila quadrilineata with a unique male behavioral element, abdomen bending

This paper describes courtship of Drosophila quadrilineata of the immigrans group and reports a species-specific element, abdomen bending, that has not been described in Drosophila. The male bends his abdomen right and left as a display to the female. During courtship the male circles around the female, bends his abdomen, and taps her. The male licks the female ovipositor repeatedly, often after the females extrusion. Although the females ovipositor extrusion is thought to indicate rejection of a courting male in many species, the male of D. quadrilineata continues courtship. It is possible that the extrusion is a females mating signal to the courting male. The male of D. quadrilineata mounts the female in a rearward position and the genitalia of both sexes couple after mounting. This mounting position is shared by the species belonging to the immigrans group. We never observed behavior in which it appeared as if the male displayed the black stripes to the female. Males court one another and a courtship chain is frequently formed even in the absence of females. The males abdomen bending and the females extrusion followed by the males licking are discussed.     

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.