Rhomboid function in the midline of the Drosophila CNS

Rhomboid (Rho), a cell surface, seven-transmembrane domain protein, participates in Spitz-dependent activation of the Drosophila EGF receptor (EGFR). By contrast to transient expression in other embryonic tissues, rho is expressed continuously in the embryonic and larval Midline Glia (MG) lineage and is required upstream of, or in parallel with, S, Spi, and EGFR to establish MG cell number. EGFR signaling is necessary for the expression of rho in the MG and sufficient to stimulate rho expression in additional MG progenitors. rho expression is required continuously from embryonic stage 9-17 to suppress apoptosis in the MG. Although rho misexpression can increase MG number through a non-cell autonomous mechanism, the pattern of normal rho expression suggests that it functions by enhancing autocrine or paracrine signaling among MG cells.     

Rhomboid 3 orchestrates Slit-independent repulsion of tracheal branches at the CNS midline

EGF-receptor ligands act as chemoattractants for migrating epithelial cells during organogenesis and wound healing. We present evidence that Rhomboid 3/EGF signalling, which originates from the midline of the Drosophila ventral nerve cord, repels tracheal ganglionic branches and prevents them from crossing it. rho3 acts independently from the main midline repellent Slit, and originates from a different sub-population of midline cells: the VUM neurons. Expression of dominant-negative Egfr or Ras induces midline crosses, whereas activation of the Egfr or Ras in the leading cell of the ganglionic branch can induce premature turns away from the midline. This suggests that the level of Egfr intracellular signalling, rather than the asymmetric activation of the receptor on the cell surface, is an important determinant in ganglionic branch repulsion. We propose that Egfr activation provides a necessary switch for the interpretation of a yet unknown repellent function of the midline.     

Axon pathfinding proceeds normally despite disrupted growth cone decisions at CNS midline

Axons in the bilateral brain of Drosophila decide whether or not to cross the midline before following their specific subsequent pathways. In commissureless mutants, the RP3 and V motoneuron axons often fail to cross the midline but subsequently follow the mirror-image pathways and innervate corresponding muscle targets on the ipsilateral side. Conversely, in roundabout mutants, the RP2 and aCC motoneuron axons sometimes cross the midline abnormally but their subsequent pathways and synaptic targeting are the perfect mirror images of those seen in wild type. Furthermore, within a single segment of these mutants, bilateral pairs of motoneuron axons can make their midline decisions independently of each other. Thus, neither the growth cones' particular molecular experience nor the decision at the midline caused by these mutations affects their ability to respond normally to subsequently presented cues.     

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.     

Drosophila single-minded gene and the molecular genetics of CNS midline development.

Our goal is to understand the molecular mechanisms that govern the formation of the central nervous system. In particular, we have focused on the development of a small group of neurons and glia that lie along the midline of the Drosophila CNS. These midline cells possess a number of unique attributes which make them particularly amenable to molecular, cellular, and genetic examinations of nervous system formation and function. In addition, the midline cells exhibit distinctive ontogeny, morphology, anatomical position, and patterns of gene expression which suggest that they may provide unique functions to the developing CNS. The single-minded gene encodes a nuclear protein which is specifically expressed in the midline cells and has been shown to play a crucial role in midline cell development and CNS formation. Genetic experiments reveal that sim is required for the expression of many CNS midline genes which are thought to be involved in the proper differentiation of these cells. In order to identify additional genes which are expressed in some or all of the midline cells at different developmental stages, a technique known as enhancer trap screening was employed. This screen led to the identification of a large number of potential genes which exhibit various midline expression patterns and may be involved in discrete aspects of midline cell development. Further molecular, genetic, and biochemical analyses of sim and several of the enhancer trap lines are being pursued. This should permit elucidation of the genetic hierarchy which acts in the specification, differentiation, and function of these CNS midline cells.     

Axon guidance at the midline: from mutants to mechanisms

How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Understanding the mechanisms that mediate axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for therapeutic approaches to developmental brain disorders and nerve regeneration. Achieving this understanding will require unraveling the molecular logic that ensures the proper expression and localization of axon guidance cues and receptors, and elucidating the signaling events that regulate the growth cone cytoskeleton in response to guidance receptor activation. Studies of axon guidance at the midline of many experimental systems, from the ventral midline of Drosophila to the vertebrate spinal cord, have led to important mechanistic insights into the complex problem of wiring the nervous system. Here we review recent advances in understanding the regulation of midline axon guidance, with a particular emphasis on the contributions made from molecular genetic studies of invertebrate model systems.     

CNS midline cells contribute to maintenance of the initial dorsoventral patterning of the Drosophila ventral neuroectoderm

Dorsoventral patterning of the Drosophila ventral neuroectoderm is established by the expression of three evolutionarily conserved homeodomain genes: ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) in the medial, intermediate, and lateral columns of the ventral neuroectoderm, respectively. It was not clear whether extrinsic factor(s) from the CNS midline cells influence the initial dorsoventral patterning by controlling the expression of the dorsoventral patterning genes. We show here that the CNS midline cells, specified by single-minded (sim), are essential for maintaining expression of the dorsoventral patterning genes. Ectopic expression of sim in the ventral neuroectoderm during the blastoderm stage repressed expression of the three homeodomain genes in the ventral neuroectoderm. This indicates that the identity of the CNS midline cells is established by a series of repressions of the three homeodomain genes in the ventral neuroectoderm. Ectopic expression of sim in the ventral neuroectoderm during initial neurogenesis induced ectopic ind expression in the medial column in addition to that in the intermediate column via EGFR signaling between the ventral neuroectoderm and midline cells. In contrast, it repressed the expression of vnd and msh in the medial and lateral columns, respectively. Our findings demonstrate that the CNS midline cells provide extrinsic positional information via EGFR signaling that maintains the initial subdivision of the ventral neuroectoderm into three dorsoventral columns during initial neurogenesis.     

Axon guidance: ephrins at WRK on the midline

Recent findings indicate that the embryonic motor neurons act as gatekeepers to regulate midline crossing during development of the nematode Caenorhabditis elegans. The newly identified protein WRK-1 and ephrins cooperate to prevent longitudinal axons from crossing the midline.     

CNS midline cells are required for establishment and differentiation of Drosophila MP2 interneurons

The Drosophila CNS develops from the ventral neuroectoderm (VNE) on both sides of the midline along the dorsoventral axis. During early neurogenesis, three homeodomain and Egfr signaling genes are required for the dorsoventral patterning of the VNE. However, the roles of CNS midline cells in patterning of the specific neural lineages are not well understood. Their roles in identity determination and differentiation of the well-established MP2 lineage were studied using several molecular markers. We showed that these cells are essential for identity determination of the MP2 lineage that originates from the VNE. The midline cells and the Egfr signaling genes were also required for the proper maintenance of MP2 and the correct formation of MP2 axonal pathways. Overexpression of sim in the midline cells activated ectopic expression of MP2 markers in the VNE. This analysis suggests that CNS midline cells and Egfr signaling genes play essential roles in the proper establishment and differentiation of the MP2 lineage.     

Slit stimulation recruits Dock and Pak to the roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.     

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     

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.