Regulation of the Drosophila dopa decarboxylase gene in neuronal and glial cells

A cis-regulatory element selectively required for the Drosophila melanogaster dopa decarboxylase gene (Ddc) in the central nervous system has been identified previously (Scholnick et al. 1986). Here, we show that at least one additional regulatory element is required for normal neuronal expression of Ddc. We find that Ddc is normally expressed in about 125 discrete neurons and in a diffused network comprising a subset of glial cells. The expression of in vitro-altered Ddc genes was studied by immunohistochemistry following germ line reintegration with P-element vectors. Normal neuron-specific Ddc gene expression requires both the initially identified element (element I) which is 60 bp upstream from the RNA start site, and an additional regulatory element located 800-2200 bp upstream. This latter element is required for neuronal expression but is not necessary for glial expression of Ddc. We provide a model to explain how interactions between multiple regulatory elements may serve to specify cell-specific gene expression.     

Two checkpoint complexes are independently recruited to sites of DNA damage in vivo

The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.     

The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast

DDC2 is a novel component of the DNA integrity checkpoint pathway, which is required for proper checkpoint response to DNA damage and to incomplete DNA replication. Moreover, Ddc2 overproduction causes sensitivity to DNA-damaging agents and checkpoint defects. Ddc2 physically interacts with Mec1 and undergoes Mec1-dependent phosphorylation both in vitro and in vivo. The phosphorylation of Ddc2 takes place in late S phase and in G(2) phase during an unperturbed cell cycle and is further increased in response to DNA damage. Because Ddc2 phosphorylation does not require any other known tested checkpoint factors but Mec1, the Ddc2-Mec1 complex might respond to the presence of some DNA structures independently of the other known checkpoint proteins. Our findings suggest that Ddc2 may be the functional homolog of Schizosaccharomyces pombe Rad26, strengthening the hypothesis that the mechanisms leading to checkpoint activation are conserved throughout evolution.     

A neuron-specific enhancer of the Drosophila dopa decarboxylase gene

At least two cis-regulatory elements are necessary for correct neuron-specific expression of the Drosophila melanogaster dopa decarboxylase gene, Ddc. In addition to a previously described proximal element located approximately 60 bp upstream of the mRNA start site, we have now characterized a distal approximately 600-bp DNA fragment, extending from -1019 to -1623 bp, which possesses enhancer-like properties and is essential for normal neuron-specific expression. Immunofluorescent labeling of neurons expressing deleted Ddc genes indicates that this region contains both general neuronal regulatory elements and cell-specific elements that selectively affect Ddc expression in either dopaminergic or serotonergic neurons. These selective effects can be correlated with the removal of sequence elements that are protected from DNase digestion by factors present in embryonic nuclear extracts. Several of these elements are also homologous to sequences located upstream of the evolutionarily diverged Ddc gene of Drosophila virilis. These results suggest that the neuron-specific expression of Ddc results from the combined action of several factors binding within this distal enhancer region.     

Species-specific patterns of sexual dimorphism in the expression of fruitless protein, a neural musculinizing factor in Drosophila

In Drosophila melanogaster, male-specific forms of the fruitless (fru) gene product, mFru protein, function as a neural sex-determination factors that directs the development of at least two male characteristics, namely courtship and mating behavior and the formation of the muscle of Lawrence (MOL). In D. melanogaster, the male-specific expression of Fru protein in motoneurons is responsible for the male-limited induction of the MOL by such neurons. Although no Drosophila species whose females have the MOL are known, there are many Drosophila species whose males lack the MOL. We performed immunohistochemical staining of the central nervous system (CNS) from 9 Drosophila species to determine whether the mFru expression profile is different between MOL-present and MOL-absent species. In 8 of the 9 species, Fru protein expression in the CNS is strictly male-specific, regardless of the presence or absence of the MOL. The sole exception is D. suzukii, in which females express the Fru protein though less extensively than males do: Fru expression in the CNS of female D. suzukii is restricted to the lamina and ventral ganglia. Expression of Fru protein in the lamina is observed in males of D. virilis and in both sexes of D. suzukii, but not in males and females of the 7 other species. These results indicate that sexually dimorphic expression of the Fru protein has been subjected to species-specific modulation during evolution.     

Prox1 expression patterns in the developing and adult murine brain.

Prox1, a homeobox gene related to the Drosophila gene prospero, is necessary for retina, lens, liver, pancreas, and lymphatics development. However, not much is yet known about Prox1 expression during central nervous system development. Here we provide a detailed analysis of Prox1 mRNA and protein expression during prenatal and postnatal murine brain development. Prenatally, Prox1 is expressed in the subventricular zone or in early differentiating regions of the brain. At these stages, Prox1 mRNA, but not Prox1 protein, was also detected in several regions of the prethalamus and hypothalamus. At an early postnatal stage, Prox1 expression is mainly detected in several nuclei of the thalamus, the cerebellum, and the hippocampus. In adulthood, Prox1 expression remains only in the hippocampus and cerebellum. These complex patterns of expression suggest that Prox1 activity is differentially required during brain development and adulthood.     

A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint

The pachytene checkpoint prevents meiotic cell cycle progression in response to unrepaired recombination intermediates. We show that Ddc1 is required for the pachytene checkpoint in Saccharomyces cerevisiae. During meiotic prophase, Ddc1 localizes to chromosomes and becomes phosphorylated; these events depend on the formation and processing of double-strand breaks (DSBs). Ddc1 colocalizes with Rad51, a DSB-repair protein, indicating that Ddc1 associates with sites of DSB repair. The Rad24 checkpoint protein interacts with Ddc1 and with recombination proteins (Sae1, Sae2, Rad57, and Msh5) in the two-hybrid protein system, suggesting that Rad24 also functions at DSB sites. Ddc1 phosphorylation and localization depend on Rad24 and Mec3, consistent with the hypothesis that Rad24 loads the Ddc1/Mec3/Rad17 complex onto chromosomes. Phosphorylation of Ddc1 depends on the meiosis-specific kinase Mek1. In turn, Ddc1 promotes the stable association of Mek1 with chromosomes and is required for Mek1-dependent phosphorylation of the meiotic chromosomal protein Red1. Ddc1 therefore appears to operate in a positive feedback loop that promotes Mek1 function.     

Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo

BACKGROUND: The mammalian receptor protein tyrosine kinase (RTK), Anaplastic Lymphoma Kinase (ALK), was first described as the product of the t(2;5) chromosomal translocation found in non-Hodgkin's lymphoma. While the mechanism of ALK activation in non-Hodgkin's lymphoma has been examined, to date, no in vivo role for this orphan insulin receptor family RTK has been described. RESULTS: We describe here a novel Drosophila melanogaster RTK, DAlk, which we have mapped to band 53 on the right arm of the second chromosome. Full-length DAlk cDNA encodes a phosphoprotein of 200 kDa, which shares homology not only with mammalian ALK but also with the orphan RTK LTK. Analysis of both mammalian and Drosophila ALK reveals that the ALK family of RTKs contains a newly identified MAM domain within their extracellular domains. Like its mammalian counterpart, DAlk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of DAlk in the Drosophila brain, careful analysis reveals an additional early role for DAlk in the developing visceral mesoderm where its expression is coincident with activated ERK. CONCLUSION: In this paper we describe a Drosophila melanogaster Alk RTK which is expressed in the developing embryonic mesoderm and CNS. Our data provide evidence for the existence of a DAlk RTK pathway in Drosophila. We show that ERK participates in this pathway, and that it is activated by DAlk in vivo. Expression patterns of dALK, together with activated ERK, suggest that DAlk fulfils the criteria of the missing RTK pathway, leading to ERK activation in the developing visceral mesoderm.     

Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes

Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte-surface-associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine-derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes.     

Elf-1在非小细胞肺癌中的表达与肿瘤细胞凋亡及患者预后的关系

目的探讨转录因子Elf-1在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达与凋亡抑制蛋白(inhibitorof apoptosis protein,IAP)家族中最强的凋亡抑制因子survivin关系。方法采用免疫组化PV-9000法检测60例NSCLC组织芯片及9例正常肺组织中Elf-1的表达水平;脱氧核苷酸移换酶(terminal deoxynucleotidy transferase,TdT)介导的dUTP切口末端标记方法(TUNEL)检测癌细胞凋亡指数(apoptosis index,AI)。结果在正常肺组织中有1例Elf-1弱阳性,余为阴性,在NSCLC组织中的阳性率为70.0%,其表达水平与肿瘤细胞分化程度、淋巴结转移、临床分期和术后生存期有关(P<0.05)。Kaplan-Meier生存分析表明其过表达与患者的生存率有关,阳性患者的生存率明显低于阴性患者(P<0.01)。Spearman相关分析显示,Elf-1在NSCLC组织中的表达与AI呈负相关(r=-0.708,P<0.01)。结论 Elf-1在NSCLC组织中的过表达影响着肿瘤细胞的凋亡,与肿瘤细胞分化程度、淋巴结转移和预后有关,检测其表达水平可作为判定NSCLC恶性生物学行为的参考指标。     

转录因子 Elf-1在上皮性卵巢癌中的表达及其意义

目的 探讨转录因子Elf-1在上皮性卵巢癌中的表达及其临床意义,并探讨Elf-1与卵巢癌细胞增殖的相关性.方法 收集31例上皮性卵巢肿瘤组织及3例正常卵巢组织标本,采用免疫组化S-P法测定Elf-1在上皮性卵巢癌中的表达并对其表达情况进行分析.结果 在23例上皮性卵巢癌组织中Elf-1阳性表达15例(65.22%).Elf-1的表达与组织学类型有关(χ2=6.54,P＜0.05),与患者的年龄、FIGO临床分期差异无统计学意义(χ2=0.25和0.08,P＞0.05).Elf-1在上皮性卵巢癌中的表达与c-Fos在上皮性卵巢癌中的表达呈正相关(Kappar=0.39,P＞0.05).结论 Elf-1在上皮性卵巢癌组织中呈高水平表达,且与原癌基因c-Fos的表达呈正相关,提示Elf-1可能通过影响上皮性卵巢癌细胞的增殖而参与其发生和发展.     

转录因子Elf-1在上皮性卵巢癌中的表达及其意义

目的探讨转录因子Elf-1在上皮性卵巢癌中的表达及其临床意义,并探讨Elf-1与卵巢癌细胞增殖的相关性。方法收集31例上皮性卵巢肿瘤组织及3例正常卵巢组织标本,采用免疫组化S-P法测定Elf-1在上皮性卵巢癌中的表达并对其表达情况进行分析。结果在23例上皮性卵巢癌组织中Elf-1阳性表达15例（65．22％）。Elf-1的表达与组织学类型有关（χ2=6．54,P〈0．05）,与患者的年龄、FIGO临床分期差异无统计学意义（χ2=0．25和0．08,P〉0．05）。Elf-1在上皮性卵巢癌中的表达与c-Fos在上皮性卵巢癌中的表达呈正相关（Kappar=0．39,P〉0．05）。结论Elf-1在上皮性卵巢癌组织中呈高水平表达,且与原癌基因c-Fos的表达呈正相关,提示Elf-1可能通过影响上皮性卵巢癌细胞的增殖而参与其发生和发展。     

The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization

In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. Here, we show that the gene kette is required for the normal projection of the VUM axons and subsequently disrupts glial migration. Axonal projection defects are also found for many other moto- and interneurons. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The KETTE protein is homologous to the transmembrane protein HEM-2/NAP1 evolutionary conserved from worms to vertebrates. In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, we show that kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins RAC1 and CDC42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant DCDC42 or DRAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated DRAC1 transgene. Our data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding.     

Checkpoint kinases and the INO80 nucleosome remodeling complex enhance global chromatin mobility in response to DNA damage

Double-strand break repair by recombination requires a homology search. In yeast, induced breaks move significantly more than undamaged loci. To examine whether DNA damage provokes an increase in chromatin mobility generally, we tracked undamaged loci under DNA-damaging conditions. We found that the yeast checkpoint factors Mec1, Rad9, and Rad53 are required for genome-wide increases in chromatin mobility, but not the repair protein Rad51. Mec1 activation by targeted Ddc1/Ddc2 enhances chromatin mobility even in the absence of damage. Finally, the INO80 chromatin remodeler is shown to act downstream from Mec1 to increase chromatin mobility, highlighting an additional damage-related role of this nucleosome remodeling complex.     

PLEXIN-D1, a novel plexin family member, is expressed in vascular endothelium and the central nervous system during mouse embryogenesis.

The genetic defect in M?bius syndrome 2 (MBS2, MIM 601471), a dominantly inherited disorder characterised by paralysis of the facial nerve, is situated at chromosome 3q21-q22. We characterised the cDNA and predicted protein, and examined the expression pattern during mouse embryogenesis of a positional candidate gene, PLEXIN-D1 (PLXND1). The cDNA for PLXND1 is 7095 base pairs in length, coding for a predicted protein of 1925 amino acids. The protein features all known domains of plexin family members, with the exception of the third Met-related sequence. Northern analysis revealed a very low expression of PLXND1 in adult mouse and adult human tissues. To investigate the expression of PlxnD1 during embryogenesis, RNA in situ hybridisation was performed on mouse embryos from various stages. This investigation revealed expression of PlxnD1 in cells from the central nervous system (CNS) and in vascular endothelium. Early expression in the CNS is located in the ganglia, cortical plate of the cortex, and striatum. At later embryologic stages, neural expression was also seen in the external granular layer of the cerebellum and several nerve nuclei. The expression in the vascular system resides solely in the endothelial cells of developing blood vessels. Based on our results, we suggest that this expression of a member of the plexin family in vascular endothelium could point toward a role in embryonic vasculogenesis.