敲低piwi基因对黑腹果蝇血细胞增殖及分化的影响

【目的】探究敲低piwi基因对黑腹果蝇Drosophila melanogaster血细胞增殖及分化的影响。【方法】利用黑腹果蝇e33C-Gal4和Hml-Gal4-UAS-2×EGFP品系分别与野生型w¹¹¹⁸和UAS-piwi RNAi品系杂交,实现在黑腹果蝇游离血细胞或淋巴腺中降低piwi基因的表达;采用免疫荧光染色方法检测Piwi蛋白在血细胞中的定位及其对黑腹果蝇血细胞增殖与分化的影响。【结果】Piwi蛋白在黑腹果蝇游离血细胞及整个淋巴腺中都表达,且主要定位在细胞质;敲低piwi基因导致游离血细胞数量明显增加,处于有丝分裂M期的细胞数量增加,但未影响游离血细胞中浆细胞及薄层细胞的分化;敲低piwi基因对淋巴腺血细胞增殖无影响,但导致浆细胞过度分化及薄层细胞的产生。【结论】piwi基因在果蝇游离血细胞中的缺失可引起血细胞过度增殖,而在淋巴腺中敲低可引起血细胞的异常分化。     

Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development

To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.     

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.     

Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis

BACKGROUND: During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. RESULTS: We show here that the mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA(37), Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA(37) mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, we show that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. CONCLUSIONS: Our results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells.     

Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the <Emphasis Type="Italic">Drosophila</Emphasis> lymph gland

The Drosophila melanogaster hematopoietic organ, called lymph gland, proliferates and differentiates throughout the larval period. The lymph gland of the late larva is comprised of a large primary lobe and several smaller secondary lobes. Differentiation into two types of hemocytes, plasmatocytes and crystal cells, is confined to the outer layer (cortical zone) of the primary lobe; the center of the primary lobe (medullary zone), as well as the secondary lobes, contain only proliferating prohemocytes. A small cluster of cells located at the posterior tip of the primary lobe serves as a signaling center (PSC) that inhibits precocious differentiation of the medullary zone. The larval lymph gland is stabilized by layers of extracellular matrix (basement membranes) that surround individual hemocytes, groups of hemocytes, as well as the lymph gland as a whole. In this paper, we investigated the events shaping the lymph gland in the early pupa. The lymph gland dissociates and hemocytes disperse during the first 12 h after puparium formation (APF), leaving behind empty husks of basement membrane. Prior to lymph gland dissociation, cells of the medullary zone differentiate, expressing the early differentiation marker Peroxidasin (Pxn), as well as, in part, the late differentiation marker P1. Cells of the PSC spread throughout the pupal lymph gland prior to their dispersal. Cells of the secondary lobes undergo a rapid phase of proliferation that lasts until 8 h APF, followed by expression of Pxn and dispersal. These hemocytes do not express P1, indicating that they disperse prior to full maturation.     

Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.     

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk(-/-) overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence that Src activity requires JNK and STAT function.     

The Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, we define the developmental steps in the maturation of blood cells (hemocytes) from their precursors. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis.     

Requirement of RBP9, a Drosophila Hu homolog, for regulation of cystocyte differentiation and oocyte determination during oogenesis

The Drosophila RNA binding protein RBP9 and its Drosophila and human homologs, ELAV and the Hu family of proteins, respectively, are highly expressed in the nuclei of neuronal cells. However, biochemical studies suggest that the Hu proteins function in the regulation of mRNA stability, which occurs in the cytoplasm. In this paper, we show that RBP9 is expressed not only in the nuclei of neuronal cells but also in the cytoplasm of cystocytes during oogenesis. Despite the predominant expression of RBP9 in nerve cells, mutational analysis revealed a female sterility phenotype rather than neuronal defects for Rbp9 mutants. The female sterility phenotype of the Rbp9 mutants resulted from defects in oogenesis; the lack of Rbp9 activity caused the germarium region of the mutants to be filled with undifferentiated cystocytes. RBP9 appears to stimulate cystocyte differentiation by regulating the expression of bag-of-marbles (bam) mRNA, which encodes a developmental regulator of germ cells. RBP9 protein bound specifically to bam mRNA in vitro, which is required for cystocyte proliferation, and the number of cells that expressed BAM protein was increased 5- to 10-fold in the germarium regions of Rbp9 mutants. These results suggest that RBP9 protein binds to bam mRNA to down regulate BAM protein expression, which is essential for the initiation of cystocyte differentiation into functional egg chambers. In hypomorphic Rbp9 mutants, cystocytes differentiated into egg chambers; however, oocyte determination and positioning were perturbed. Therefore, the concentrated localization of RBP9 protein in the oocyte of the early egg chambers may be required for proper oocyte determination or positioning.     

果蝇spen蛋白的抗体制备、组织特异性表达及功能分析

Spen家族蛋白参与多种生物学过程,包括神经元细胞的命运、神经元突起延伸的调节、细胞周期调控等,并且是联系Notch信号途径和生长因子受体途径的关键分子。最近的研究表明spen基因在果蝇的眼睛、翅膀和腿组织中参与Wnt信号转导。但该基因在果蝇中的功能还有很多不明确之处。文章采用基因克隆、原核表达及亲和层析等方法制备并纯化了黑腹果蝇spen的C端6×His-spen融合蛋白,以纯化的融合蛋白免疫大鼠获得了抗spen的多克隆抗体。利用制备的抗体进行免疫染色结果显示spen蛋白定位于细胞核内,并且在大脑、脂肪体、血细胞、肠和唾液腺等组织中表达量较高。分析野生型和突变体果蝇血细胞的噬菌作用,发现spen蛋白低表达的突变体吞噬外来异物明显低于野生型,结果表明spen蛋白能够调节血细胞的吞噬功能。     

The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.     

The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland

The heparin sulfate proteoglycan Terribly Reduced Optic Lobes (Trol) is the Drosophila melanogaster homolog of the vertebrate protein Perlecan. Trol is expressed as part of the extracellular matrix (ECM) found in the hematopoietic organ, called the lymph gland. In the normal lymph gland, the ECM forms thin basement membranes around individual or small groups of blood progenitors. The pattern of basement membranes, reported by Trol expression, is spatio-temporally correlated to hematopoiesis. The central, medullary zone which contain undifferentiated hematopoietic progenitors has many, closely spaced membranes. Fewer basement membranes are present in the outer, cortical zone, where differentiation of blood cells takes place. Loss of trol causes a dramatic change of the ECM into a three-dimensional, spongy mass that fills wide spaces scattered throughout the lymph gland. At the same time proliferation is reduced, leading to a significantly smaller lymph gland. Interestingly, differentiation of blood progenitors in trol mutants is precocious, resulting in the break-down of the usual zonation of the lymph gland. which normally consists of an immature center (medullary zone) where cells remain undifferentiated, and an outer cortical zone, where differentiation sets in. We present evidence that the effect of Trol on blood cell differentiation is mediated by Hedgehog (Hh) signaling, which is known to be required to maintain an immature medullary zone. Overexpression of hh in the background of a trol mutation is able to rescue the premature differentiation phenotype. Our data provide novel insight into the role of the ECM component Perlecan during Drosophila hematopoiesis.     

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.     

Of blood cells and the nervous system

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.     

Drosophila ribosomal protein mutants control tissue growth non-autonomously via effects on the prothoracic gland and ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycE(JP)). We demonstrated that the suppression of cycE(JP) by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycE(JP) is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.     

Of blood cells and the nervous system: Hematopoiesis in the Drosophila larva

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.