Neurogenesis in the spider Cupiennius salei.

To uncover similarities and differences in neurogenesis in arthropod groups, we have studied the ventral neuroectoderm of the spider Cupiennius salei (Chelicerata, Aranea, Ctenidae). We found that invaginating cell groups arose sequentially, at stereotyped positions in each hemisegment and in separate waves, comparable with the generation of neuroblasts in Drosophila. However, we found no evidence for proliferating stem cells that would be comparable with the neuroblasts. Instead, the whole group of invaginating cells was directly recruited to the nervous system. The invagination process is comparable with Drosophila, with the cells attaining a bottle-shaped form with the nuclei moving inwards, while actin-rich cell processes remain initially connected to the surface of the epithelium. This general pattern is also found in another spider, Pholcus phalangioides, and appears thus to be conserved at least among the Araneae. We have identified two basic helix-loop-helix encoding genes -- CsASH1 and CsASH2 -- that share sequence similarities with proneural genes from other species. Functional analysis of the genes by double-stranded RNA interference revealed that CsASH1 was required for the formation of the invagination sites and the process of invagination itself, whereas CsASH2 seemed to be required for the differentiation of the cells into neurones. Our results suggest that the basic processes of neurogenesis, as well as proneural gene function is conserved among arthropods, apart of the lack of neuroblast-like stem cells in spiders.

Changes in cell shape in the ventral neuroectoderm of Drosophila melanogaster depend on the activity of the achaete-scute complex genes.

In the embryonic ventral neuroectoderm of Drosophila melanogaster the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l'sc suggest that these genes are responsible for most of the morphological alterations observed.

loco encodes an RGS protein required for Drosophila glial differentiation.

In Drosophila, glial cell development depends on the gene glial cells missing (gcm). gcm activates the expression of other transcription factors such as pointed and repo, which control subsequent glial differentiation. In order to better understand glial cell differentiation, we have screened for genes whose expression in glial cells depends on the activity of pointed. Using an enhancer trap approach, we have identified loco as such a gene. loco is expressed in most lateral CNS glial cells throughout development. Embryos lacking loco function have an normal overall morphology, but fail to hatch. Ultrastructural analysis of homozygous mutant loco embryos reveals a severe glial cell differentiation defect. Mutant glial cells fail to properly ensheath longitudinal axon tracts and do not form the normal glial-glial cell contacts, resulting in a disruption of the blood-brain barrier. Hypomorphic loco alleles were isolated following an EMS mutagenesis. Rare escapers eclose which show impaired locomotor capabilities. loco encodes the first two known Drosophila members of the family of Regulators of G-protein signalling (RGS) proteins, known to interact with the alpha subunits of G-proteins. loco specifically interacts with the Drosophila alphai-subunit. Strikingly, the interaction is not confined to the RGS domain. This interaction and the coexpression of LOCO and Galphai suggests a function of G-protein signalling for glial cell development.

Glial development in the Drosophila CNS requires concomitant activation of glial and repression of neuronal differentiation genes.

Two classes of glial cells are found in the embryonic Drosophila CNS, midline glial cells and lateral glial cells. Midline glial development is triggered by EGF-receptor signalling, whereas lateral glial development is controlled by the gcm gene. Subsequent glial cell differentiation depends partly on the pointed gene. Here we describe a novel component required for all CNS glia development. The tramtrack gene encodes two zinc-finger proteins, one of which, ttkp69, is expressed in all non-neuronal CNS cells. We show that ttkp69 is downstream of gcm and can repress neuronal differentiation. Double mutant analysis and coexpression experiments indicate that glial cell differentiation may depend on a dual process, requiring the activation of glial differentiation by pointed and the concomitant repression of neuronal development by tramtrack.

Electron microscopic analysis of Drosophila midline glia during embryogenesis and larval development using beta-galactosidase expression as endogenous cell marker.

To thoroughly study developmental problems it is often desirable to identify specific cells at the resolution of the electron microscope (TEM). Specific antibodies, and immunogold and other antibody labelling techniques can be successfully used with the TEM. But for these techniques to be successful there must be substantial adjustments for each antibody and tissue analyzed. To develop a more generally applicable labelling method we took advantage of the enhancer trap technique in Drosophila. Enhancer trap fly strains show cell- and/or tissue-specific beta-galactosidase expression which can be visualized by a simple X-gal staining procedure. To combine the power of the enhancer trap approach with electron microscopy, we have improved the fixation and staining conditions, which allow detection of X-gal crystals (by TEM) and thus provide precise information on ultrastructural morphology. We have tested our technique using the well-known midline glial cells and examined these cells between late embryonic and pupal developmental stages. The four embryonic midline glial cells found in each neuromere reside ventrally and dorsally to the midline of the neuropile and are closely associated with unpaired neurons, major commissures, and other types of glial cells. During larval and pupal life dramatic cell growth and endomitotic nuclear replication occur in midline glial cells. By the end of larval life, the giant midline glial cells fragment to give rise to a variable number of small midline glial cells. Here we show that the combination of transmission electron microscopy with cytochemical detection of beta-galactosidase expression represents a promising and valuable tool for the study of the morphology and development of specific cell types.

Development and function of embryonic central nervous system glial cells in Drosophila.

Each abdominal neuromere of a Drosophila embryo contains about 60 glial cells [Klämbt C, Goodman CS (1991): Glia 4:205-213; Ito et al. (1995): Roux's Arch Dev Biol, 204:284-307]. Among these, the midline and longitudinal glia are described to some detail. The midline glia are located dorsally in the nerve cord ensheathing the two segmental commissures. They are required for the proper establishment of commissures. The longitudinal glia, the A and B glia, and the segment boundary cells (SBC) are covering the longitudinal connectives. The longitudinal glia prefigure longitudinal axon paths and appear capable of regulating the expression of neuronal antigens. In the following we summarize the knowledge on the function of these glial cells.

The Ets transcription factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic CNS.

The Drosophila gene pointed (pnt) encodes two putative transcription factors (P1 and P2) of the Ets family, which in the embryonic CNS are found exclusively in glial cells. Loss of pnt function leads to poorly differentiated glial cells and a marked decrease in the expression of the neuronal antigen 22C10 in the MP2 neurons, which are known to interact intimately with the pntP1-expressing longitudinal glial cells. Ectopic expression of pntP1 RNA forces additional CNS cells to enter the glial differentiation pathway. Interestingly, the additional glial-like cells are often flanked by cells that ectopically express the neuronal antigen 22C10. Therefore, both the pnt loss-of-function as well as the gain-of-function phenotype suggest that glial cells are able to induce 22C10 expression on neighboring neurons. This was further verified by cell transplantation experiments. Thus, pnt is not only required but also sufficient for several aspects of glial differentiation.