Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252.

During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In our study we show that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. We determined the abundance of the two different otp transcripts in the brain and hindgut during development using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. We generated an otp enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, we looked for putative miRNAs interacting with the otp 3'UTR, and identified microRNA-252 as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3'UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3'UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. Our results show that Otp is another important factor for brain development in Drosophila melanogaster.

Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development.

BACKGROUND: The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. RESULTS: In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. CONCLUSION: The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

Enhancer analysis of the Drosophila zinc finger transcription factor Earmuff by gene targeting.

BACKGROUND: Many transcription factors are involved in the formation of the brain during the development of Drosophila melanogaster. The transcription factor Earmuff (Erm), a member of the forebrain embryonic zinc finger family (Fezf), is one of these important factors for brain development. One major function of Earmuff is the regulation of proliferation within type II neuroblast lineages in the brain; here, Earmuff is expressed in intermediate neural progenitor cells (INPs) and balances neuronal differentiation versus stem cell maintenance. Erm expression during development is regulated by several enhancers. RESULTS: In this work we show a functional analysis of erm and some of its enhancers. We generated a new erm mutant allele by gene targeting and reintegrated Gal4 to make an erm enhancer trap strain that could also be used on an erm mutant background. The deletion of three of the previously analysed enhancers showing the most prominent expression patterns of erm by gene targeting resulted in specific temporal and spatial defects in defined brain structures. These defects were already known but here could be assigned to specific enhancer regions. CONCLUSION: This analysis is to our knowledge the first systematic analysis of several large enhancer deletions of a Drosophila gene by gene targeting and will enable deeper analysis of erm enhancer functions in the future.

Functional analysis of enhancer elements regulating the expression of the Drosophila homeodomain transcription factor DRx by gene targeting.

BACKGROUND: The Drosophila brain is an ideal model system to study stem cells, here called neuroblasts, and the generation of neural lineages. Many transcriptional activators are involved in formation of the brain during the development of Drosophila melanogaster. The transcription factor Drosophila Retinal homeobox (DRx), a member of the 57B homeobox gene cluster, is also one of these factors for brain development. RESULTS: In this study a detailed expression analysis of DRx in different developmental stages was conducted. We show that DRx is expressed in the embryonic brain in the protocerebrum, in the larval brain in the DM and DL lineages, the medulla and the lobula complex and in the central complex of the adult brain. We generated a DRx enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the endogenous expression of DRx. With the help of eight existing enhancer-Gal4 strains and one made by our group, we mapped various enhancers necessary for the expression of DRx during all stages of brain development from the embryo to the adult. We made an analysis of some larger enhancer regions by gene targeting. Deletion of three of these enhancers showing the most prominent expression patterns in the brain resulted in specific temporal and spatial loss of DRx expression in defined brain structures. CONCLUSION: Our data show that DRx is expressed in specific neuroblasts and defined neural lineages and suggest that DRx is another important factor for Drosophila brain development.

Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C.

Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.

Generation of Mutants from the 57B Region of Drosophila melanogaster.

The 57B region of Drosophila melanogaster includes a cluster of the three homeobox genes orthopedia (otp), Drosophila Retinal homeobox (DRx), and homeobrain (hbn). In an attempt to isolate mutants for these genes, we performed an EMS mutagenesis and isolated lethal mutants from the 57B region, among them mutants for otp, DRx, and hbn. With the help of two newly generated deletions from the 57B region, we mapped additional mutants to specific chromosomal intervals and identified several of these mutants from the 57B region molecularly. In addition, we generated mutants for CG15651 and RIC-3 by gene targeting and mutants for the genes CG9344, CG15649, CG15650, and ND-B14.7 using the CRISPR/Cas9 system. We determined the lethality period during development for most isolated mutants. In total, we analysed alleles from nine different genes from the 57B region of Drosophila, which could now be used to further explore the functions of the corresponding genes in the future.

The Drosophila homeodomain transcription factor Homeobrain is involved in the formation of the embryonic protocerebrum and the supraesophageal brain commissure.

During the embryonic development of Drosophila melanogaster many transcriptional activators are involved in the formation of the embryonic brain. In our study we show that the transcription factor Homeobrain (Hbn), a member of the 57B homeobox gene cluster, is an additional factor involved in the formation of the embryonic Drosophila brain. Using a Hbn antibody and specific cell type markers a detailed expression analysis during embryonic brain development was conducted. We show that Hbn is expressed in several regions in the protocerebrum, including fibre tract founder cells closely associated with the supraesophageal brain commissure and also in the mushroom bodies. During the formation of the supraesophageal commissure, Hbn and FasII-positive founder cells build an interhemispheric bridge priming the commissure and thereby linking both brain hemispheres. The Hbn expression is restricted to neural but not glial cells in the embryonic brain. In a mutagenesis screen we generated two mutant hbn alleles that both show embryonic lethality. The phenotype of the hbn mutant alleles is characterized by a reduction of the protocerebrum, a loss of the supraesophageal commissure and mushroom body progenitors and also by a dislocation of the optic lobes. Extensive apoptosis correlates with the impaired formation of the embryonic protocerebrum and the supraesophageal commissure. Our results show that Hbn is another important factor for embryonic brain development in Drosophila melanogaster.

In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress.

The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1(G93A) becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1(G85R). Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS.

Mia40-dependent oxidation of cysteines in domain I of Ccs1 controls its distribution between mitochondria and the cytosol.

Superoxide dismutase 1 (Sod1) is an important antioxidative enzyme that converts superoxide anions to hydrogen peroxide and water. Active Sod1 is a homodimer containing one zinc ion, one copper ion, and one disulfide bond per subunit. Maturation of Sod1 depends on its copper chaperone (Ccs1). Sod1 and Ccs1 are dually localized proteins that reside in the cytosol and in the intermembrane space of mitochondria. The import of Ccs1 into mitochondria depends on the mitochondrial disulfide relay system. However, the exact mechanism of this import process has been unclear. In this study we detail the import and folding pathway of Ccs1 and characterize its interaction with the oxidoreductase of the mitochondrial disulfide relay Mia40. We identify cysteines at positions 27 and 64 in domain I of Ccs1 as critical for mitochondrial import and interaction with Mia40. On interaction with Mia40, these cysteines form a structural disulfide bond that stabilizes the overall fold of domain I. Although the cysteines are essential for the accumulation of functional Ccs1 in mitochondria, they are dispensable for the enzymatic activity of cytosolic Ccs1. We propose a model in which the Mia40-mediated oxidative folding of domain I controls the cellular distribution of Ccs1 and, consequently, active Sod1.

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo.

Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA(i) to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA(i) phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA(i) lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.