The midline of both the invertebrate and vertebrate nervous systems have a developmentally early and unique origin, determined or induced by a close association with the mesoderm during gastrulation. In both cases, cells of the midline are determined and differentiate before more lateral cells do. In fact, they are the source of inductive signals that determine medio-lateral identity of muscle, nervous and epidermal tissues. From an embryological perspective, the midline in both organisms comprises a developmental boundary whose position must be communicated to neighbouring cells. In vertebrates, midline signals are mediated by the notocord and the floorplate of the nerve cord. In Drosophila, the equivalent signals are mediated by the mesectoderm. These signals employ conserved signal pathways that include the EGF, Hedgehog, Netrin and Slit signaling systems.

One cell type of the mesectoderm, the MG, plays a major role in signaling midline position. For this reason, the MG express many unique genes, which, when mutant, generate visible and lethal phenotypes. At this point it is possible we know about more genes expressed by the MG than any other CNS cell type (see Jacobs, 2000).



From left to right, a temporal sequence of interaction of the MG (blue) and developing commissures (brown).(adapted from Klämbt, 1991).

Early in the development of the CNS, the first axonal growth cones explore the dorsal surface of the nerve cord, and make contacts with two classes of glial cells (Jacobs and Goodman 1989). One overlies the position of the presumptive longitudinal tracts, the other the position of the anterior commissure. Axons which cross the midline interact with neuron and glial cells before they extend to make a contralateral projection. If the MG are missing, many axons extend towards the midline and do not leave (Sonnenfeld and Jacobs, 1994). Once the first axons have established a contralateral tract, some of the MG, located at the anterior boundary of the segment, will migrate dorsally and posteriorly to separate the axons into two groups: these become the anterior and posterior commissures [See figure above]. Later in development, the MG extend oligodendroglial-like processes to ensheath the commissural axons.[See lateral view below.] Ensheathing MG contribute to the Blood Brain Barrier.

The MG and Midline Axon Guidance

Embryos mutant for genes required for MG survival and differentiation have either fused axon tracts at the midline (single-minded) or fused commissures containing axons that should not be crossing the midline (D-Egfr pathway mutants). Surprisingly, there is a mutant in which the MG survive, but axons still fuse at the midline. This gene is slit, which encodes a glycoprotein secreted by the MG.

Longitudinal axon fasicles can be identified in Drosophila larvae with an antibody to the Fasciclin II protein. Normally (left) these axons do not approach the midline. If the MG are lesioned in late embryogenesis (middle), some axon tracts fuse at the midline. In larvaeof Drosophila with reduced slit function (right), lateral axon tracts cross and recross the midline.

Work in this lab, and in Corey Goodman's, has established that Slit is the midline repellent signal that prevents axons from inappropriate crossing or re-crossingof the midline. Slit is a ligand for a receptor on growth cones, called Robo (Battye et al., 1999). Slit, and its worm and vertebrate orthologs, have a novel multidomain stricture, suggestive of interaction with the cell surface, extra-cellular matrix and cell signaling (Rothberg et al., 1990). The major structural domains are a Leucine rich region (LRR) EGF domains and a Laminin-like globular or "G" domain.

Structure-Function analysis of Slit

We have determined that the LRR domain is required for repellent signaling and for binding to the putative receptor for Slit, called Robo. For instance, axon tract extablishment is disrupted when full length Slit is expressed at the midline of transgenic Drosophila (second panel). Axon pattern is normal if Slit lacking a portion of the LRR domain is expressed in the midline (third panel). However, repellent signals from Slit are potentiated if forms of Slit with deletions in the EGF or G domains are expressed in the midline (right panels).We are continuing our characterisation of Slit function and signaling by identifying other proteins that associate with Slit in vivo.