Even-skipped, acting as a repressor, regulates axonal projections in Drosophila

Nervous system-specific eve mutants were created by removing regulatory elements from a 16 kb transgene capable of complete rescue of normal eve function. When transgenes lacking the regulatory element for either RP2+a/pCC, EL or U/CQ neurons were placed in an eve-null background, eve expression was completely eliminated in the corresponding neurons, without affecting other aspects of eve expression. Many of these transgenic flies were able to survive to fertile adulthood. In the RP2+a/pCC mutant flies: (1) both RP2 and aCC showed abnormal axonal projection patterns, failing to innervate their normal target muscles; (2) the cell bodies of these neurons were positioned abnormally; and (3) in contrast to the wild type, pCC axons often crossed the midline. The Eve HD alone was able to provide a weak, partial rescue of the mutant phenotype, while both the Groucho-dependent and -independent repressor domains contributed equally to full rescue of each aspect of the mutant phenotype. Complete rescue was also obtained with a chimeric protein containing the Eve HD and the Engrailed repressor domain. Consistent with the apparent sufficiency of repressor function, a fusion protein between the Gal4 DNA-binding domain and Eve repressor domains was capable of actively repressing UAS target genes in these neurons. A key target of the repressor function of Eve was Drosophila Hb9, the derepression of which correlated with the mutant phenotype in individual eve-mutant neurons. Finally, homologues of Eve from diverse species were able to rescue the eve mutant phenotype, indicating conservation of both targeting and repression functions in the nervous system.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

CNS midline cells influence the division and survival of lateral glia in the Drosophila nervous system

Central nervous system (CNS) midline cells are essential for identity determination and differentiation of neurons in the Drosophila nervous system. It is not clear, however, whether CNS midline cells are also involved in the development of lateral glial cells. The roles of CNS midline cells in lateral glia development were elucidated using general markers for lateral glia, such as glial cell missing and reverse polarity, and specific enhancer trap lines labeling the longitudinal, A, B, medial cell body, peripheral, and exit glia. We found that CNS midline cells were necessary for the proper expression of glial cell missing, reverse polarity, and other lateral glia markers only during the later stages of development, suggesting that they are not required for initial identity determination. Instead, CNS midline cells appear to be necessary for proper division and survival of lateral glia. CNS midline cells were also required for proper positioning of three exit glia at the junction of segmental and intersegmental nerves, as well as some peripheral glia along motor and sensory axon pathways. This study demonstrated that CNS midline cells are extrinsically required for the proper division, migration, and survival of various classes of lateral glia from the ventral neuroectoderm.     

Gene expression analysis in post-embryonic pericardial cells of Drosophila

Increasing evidence suggests conservation of cardiovascular molecules between vertebrates and invertebrates. Vertebrate Rudhira, an evolutionary conserved WD40 protein is expressed during primitive erythropoiesis, neoangiogenesis and tumors. We report here the expression profile of the Drosophila ortholog of Rudhira (DRudh) in the fly life cycle. DRudh is expressed specifically in all post-embryonic pericardial cells (PCs) and garland cells (GCs). This is the first report of a cytoplasmic marker highly specific to post-embryonic PCs. Embryonic PCs belong to three distinct genetic classes based on Odd-skipped (Odd), Even-skipped (Eve) and Tinman (Tin) expression. To identify which among these three classes of PCs expresses DRudh in post-embryonic stages, we analyzed expression of embryonic PC markers in the post-embryonic stages. Unlike in the embryo all larval PCs show an identical gene expression profile. While Odd and Eve expression is mutually exclusive in the embryonic PCs, these two markers are co-expressed in larval PCs but show a distinct subcellular localization. Tin is not expressed in any post-embryonic PC. Additionally larval PCs also express the GATA factor, Serpent (Srp) and the extracellular matrix protein, Pericardin (Prc). While PC number is known to decrease post-embryogenesis, which of the Odd or Eve lineage embryonic PCs persists is not known. Co-expression of the two distinct lineage markers only in post-embryonic stages indicates a complex temporal regulation of gene expression in PCs.     

Gon-2, a Gene Required for Gonadogenesis in Caenorhabditis Elegans

The gonad of the Caenorhabditis elegans hermaphrodite is generated by the postembryonic divisions of two somatic precursors, Z1 and Z4, and two germline precursors, Z2 and Z3. These cells begin division midway through the first larval stage. By the end of the fourth larval stage, Z1 and Z4 produce 143 descendants, while Z2 and Z3 give rise to ~1000 descendants. The divisions of Z2 and Z3 are dependent on signals produced by Z1 and Z4, but not vice versa. We have characterized the properties of five loss-of-function alleles of a newly described gene, which we call gon-2. In gon-2 mutants, gonadogenesis is severely impaired; in some animals, none of the gonad progenitors undergo any postembryonic divisions. Mutations in gon-2 have a partial maternal effect: either maternal or zygotic expression is sufficient to prevent the severe gonadogenesis defects. By cell lineage analysis, we found that the primary defect in gon-2 mutants is a delay (sometimes a complete block) in the onset and continuation of gonadal divisions. The results of upshift experiments using a temperature-sensitive allele suggest that zygotic expression of gon-2 begins early in embryogenesis, before the birth of Z1 and Z4. The results of downshift experiments suggest that Z1 and Z4 can generate the full complement of gonadal tissues even when gon-2 function is inhibited until the end of the second larval stage. Thus, gon-2 activity is probably not required for the specification of gonadal cell fates, but appears to be generally required for gonadal cell divisions.     

The zygotic control of Drosophila pair-rule gene expression. I. A search for new pair-rule regulatory loci

The examination of pair-rule gene expression in wild-type and segmentation mutant embryos has identified many, but not necessarily all, of the elements of the regulatory system that establish their periodic patterns. Here we have conducted a new type of search for previously unknown regulators of these genes by examining pair-rule gene expression in blastoderm embryos lacking parts of or entire chromosomes. This method has the advantage of direct inspection of abnormal pair-rule gene patterns without relying upon mutagenesis or interpretation of larval phenotypes for the identification of segmentation genes. From these experiments we conclude that: (i) most zygotically required regulators of the fushi tarazu (ftz), even-skipped (eve) and hairy (h) pair-rule genes have been identified, except for one or more loci we have uncovered on chromosome arm 2L; (ii) the repression of the ftz and eve genes in the anterior third of the embryo is under maternal, not zygotic control; and (iii) there are no general zygotically required activators of pair-rule gene expression. The results suggest that the molecular basis of pair-rule gene regulation can be pursued with greater confidence now that most key trans-acting factors are already in hand.     

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.     

Progressive changes in cytotoxic potential during mixed lymphocyte culture.

In a previous study it was shown that at least one round of DNA synthesis is required for initial expression of cytotoxic function in mouse lymphocytes responding to alloantigen in vitro. In the experiments reported here we ask whether subsequent rounds of cell division are required simply for clonal expansion of this initial level of cytotoxic function within the population, or whether the amount of cytotoxicity per cytotoxic cell is altered during subsequent rounds of cell division. The amount of cytotoxicity per unit number of cells at various stages of culture was compared with the frequency of cytotoxic cells as estimated principally by effector-target cell conjugates. Our results strongly suggest that the amount of cytotoxicity per cell (cytotoxic potential) is not a static property of cytotoxic cells, but can be modulated up or down during the course of a reaction.     

Regulation of membrane localization of Sanpodo by lethal giant larvae and neuralized in asymmetrically dividing cells of Drosophila sensory organs

In Drosophila, asymmetric division occurs during proliferation of neural precursors of the central and peripheral nervous system (PNS), where a membrane-associated protein, Numb, is asymmetrically localized during cell division and is segregated to one of the two daughter cells (the pIIb cell) after mitosis. numb has been shown genetically to function as an antagonist of Notch signaling and also as a negative regulator of the membrane localization of Sanpodo, a four-pass transmembrane protein required for Notch signaling during asymmetric cell division in the CNS. Previously, we identified lethal giant larvae (lgl) as a gene required for numb-mediated inhibition of Notch in the adult PNS. In this study we show that Sanpodo is expressed in asymmetrically dividing precursor cells of the PNS and that Sanpodo internalization in the pIIb cell is dependent cytoskeletally associated Lgl. Lgl specifically regulates internalization of Sanpodo, likely through endocytosis, but is not required for the endocytosis Delta, which is a required step in the Notch-mediated cell fate decision during asymmetric cell division. Conversely, the E3 ubiquitin ligase neuralized is required for both Delta endocytosis and the internalization of Sanpodo. This study identifies a hitherto unreported role for Lgl as a regulator of Sanpodo during asymmetric cell division in the adult PNS.