The jing and ras1 pathways are functionally related during CNS midline and tracheal development

The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. We show that there is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells.     

The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. Using distinct cell fate markers, we observe that in sim mutant embryos the midline cells fail to differentiate properly into their mature CNS cell types and do not take their appropriate positions within the developing CNS. We further present evidence that sim is required for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at 91F; that the sim mutant CNS defect may be largely due to loss of midline slit expression; and that the snail gene is required to repress sim and other midline genes in the presumptive mesoderm.     

Expression domains of the Cf1a POU domain protein during Drosophila development

We have used antibodies directed against a unique portion of the Drosophila POU domain protein Cfla to localize its sites of expression in developing embryos. Cfla protein is first detected during germ band extension in the tracheal placodes and in the midline mesectoderm cells. Tracheal expression continues throughout embryonic development, especially in the main longitudinal tracheal trunks. Additional sites of high Cfla expression are in the anterior portion of the hindgut, the roof of the stomodeum, a subset of central nervous system cells, the oenocytes, and the ring gland. In addition, Cfla expression was localized in embryos mutant for several loci involved in determining fate along the midline of the CNS and the tracheal system. Cfla midline cell expression is dependent on proper single-minded gene function, and Cfla either regulates or acts in parallel to the genes pointed and rhomboid during midline CNS and tracheal development.     

The CNS midline cells control the spitz class and Egfr signaling genes to establish the proper cell fate of the Drosophila ventral neuroectoderm.

The spitz class genes, pointed (pnt), rhomboid frho), single-minded (sim), spitz (spi)and Star (S), as well as the Drosophila epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis showed that the expression of all the spitz class and Egfrsignaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. It was shown that overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. On the other hand, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the EGFR signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling EGFR activity through the regulation of the expression of spitz class genes and Egfr signaling genes.     

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.     

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.     

Dissection of the faint little ball (flb) phenotype: determination of the development of the Drosophila central nervous system by early interactions in the ectoderm.

The complex embryonic phenotype of mutations in the faint little ball (flb) locus, encoding the Drosophila EGF receptor homolog (DER), was dissected by temperature shifts of a temperature-sensitive allele. We show that the phenotype can be resolved into at least five components, which are temporally and spatially distinct. Most notably, the central nervous system (CNS) phenotype is determined at two separate phases. A severe collapse results from early defects in the DER-expressing ectodermal cells from which neuroblasts and midline glial cells deaminate. We thus suggest that DER activity is crucial for interactions that occur in the ectoderm at an early stage, and determine the fate of neuronal and glial cell lineages. This finding explains how a severe CNS phenotype is generated in flb embryos, in spite of the absence of expression of the protein in neuronal cells. In a second phase, during germ band retraction, the flb function is required specifically in the three pairs of midline glial cells (MG). In the absence of a functional DER protein, these cells die or fail to differentiate correctly, resulting in a fused commissure phenotype.     

The hierarchical relationship among the spitz/Egfr signaling genes in cell fate determination in the Drosophila ventral neuroectoderm

The spitz class and Egfr signaling (spi/Egfr) genes are required for the proper establishment of cell fate in the Drosophila ventral neuroectoderm. We investigated the role of the central nervous system (CNS) midline cells, and the hierarchical relationship among the spi/Egfr genes, in this process by analyzing the spatial and temporal expression of several of the genes in selected spi/Egfr mutants. Our analysis showed that expression of all the spi/Egfr genes is severely reduced in the single-minded (sim) mutant, and ectopically induced in en-Gal4/UAS-sim embryos. This result indicates that sim acts upstream of all the other spi/Egfr genes. The CNS midline cells regulate rhomboid (rho) expression in the ventral neuroectoderm and activate the EGFR signaling pathway. We also found that argos (aos) and orthodenticle (otd) act downstream of pointed (pnt), and that aos represses expression of otd in the lateral neuroectoderm to establish differential cell fates in the ventral neuroectoderm. Our findings suggest the following hierarchical relationship among the spi/Egfr genes: [see text].     

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.     

The CNS midline cells coordinate proper cell cycle progression and identity determination of the Drosophila ventral neuroectoderm

The CNS midline cells, specified by the single-minded (sim) gene, are required for the proper patterning of the ventral CNS and epidermis, which are derived from the Drosophila ventral neuroectoderm. Defects in the sim mutant are characterized by the loss of the gene expression, which is required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defects were analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses showed that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This resulted in the loss of the dividing neuroectodermal cells that are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for the establishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. On the other hand, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that the CNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis.