SUMMARYThe lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, we characterized a PSC-active transcriptional enhancer that drives hh expression in supportive niche cells. Our findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance.

Drosophila has emerged as an important model system to discover and analyze genes controlling hematopoiesis. One regulatory network known to control hemocyte differentiation is the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signal-transduction pathway. A constitutive activation mutation of the Janus kinase Hopscotch (hopscotch(Tumorous-lethal); hop(Tum-l)) results in a leukemia-like over-proliferation of hemocytes and copious differentiation of lamellocytes during larval stages. Here we show that the Friend of GATA (FOG) protein U-shaped (Ush) is expressed in circulating and lymph gland hemocytes, where it plays a critical role in controlling blood cell proliferation and differentiation. Our findings demonstrate that a reduction in ush function results in hematopoietic phenotypes strikingly similar to those observed in hop(Tum-l) animals. These include lymph gland hypertrophy, increased circulating hemocyte concentration, and abundant production of lamellocytes. Forced expression of N-terminal truncated versions of Ush likewise leads to larvae with severe hematopoietic anomalies. In contrast, expression of wild-type Ush results in a strong suppression of hop(Tum-l) phenotypes. Taken together, our findings demonstrate that U-shaped acts to control larval hemocyte proliferation and suppress lamellocyte differentiation, likely regulating hematopoietic events downstream of Hop kinase activity. Such functions appear to be facilitated through Ush interaction with the hematopoietic GATA factor Serpent (Srp).

Drosophila has emerged as an excellent model system in which to study cellular and genetic aspects of hematopoiesis. Under normal developmental conditions and in wild-type genetic backgrounds, Drosophila possesses two types of blood cells, crystal cells and plasmatocytes. Upon infestation by a parasitic wasp or in certain altered genetic backgrounds, a third hemocyte class called the lamellocyte becomes apparent. Herein we describe the characterization of a novel transcriptional regulatory module, a lamellocyte-active enhancer of the misshapen gene. This transcriptional control sequence appears to be inactive in all cell types of the wild-type larva, including crystal cells and plasmatocytes. However, in lamellocytes induced by wasp infestation or by particular genetic conditions, the enhancer is activated and it directs reporter GFP or DsRed expression exclusively in lamellocytes. The lamellocyte control region was delimited to a 140-bp intronic sequence that contains an essential DNA recognition element for the AP-1 transcription factor. Additionally, mutation of the kayak gene encoding the dFos subunit of AP-1 led to a strong suppression of lamellocyte production in tumorous larvae. As misshapen encodes a protein kinase within the Jun N-terminal kinase signaling pathway that functions to form an active AP-1 complex, the lamellocyte-active enhancer likely serves as a transcriptional target within a genetic auto-regulatory circuit that promotes the production of lamellocytes in immune-challenged or genetically- compromised animals.

Based on environmental challenges or altered genetic composition, Drosophila larvae can produce up to three types of blood cells that express genetic programs essential for their distinct functions. Using transcriptional enhancers for genes expressed exclusively in plasmatocytes, crystal cells, or lamellocytes, several new hemocyte-specific enhancer-reporter transgenes were generated to facilitate the analysis of Drosophila hematopoiesis. This approach took advantage of fluorescent variants of insulated P-element reporter vectors for multilabeling cell analyses; two additional color variants were generated in these studies. These vectors were successfully used to produce transgenic fly lines that label specific hemocyte lineages with separate colors. Combining three transgene reporters allowed for the unambiguous identification of plasmatocytes, crystal cells, and lamellocytes within a complex hemocyte population. While this work focused on the hematopoietic process, these new vectors can be used to mark multiple cell types or trace complex cell lineages during any chosen aspect of Drosophila development.

No abstract

Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. In this report, we utilize a PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. We assessed the effect of abrogating the function of 820 genes as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, we characterized loss- and gain-of-function phenotypes. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland.

The formation of nontransmissible virus-like particles (NTVLP) by cells infected with F-deficient Sendai virus (SeV/⌬F) was found to be temperature sensitive. Analysis by hemagglutination assays and Western blotting demonstrated that the formation of NTVLP at 38°C was about 1/100 of that at 32°C, whereas this temperature-sensitive difference was only moderate in the case of F-possessing wild-type SeV. In order to reduce the NTVLP formation with the aim of improving SeV for use as a vector for gene therapy, amino acid substitutions found in temperature-sensitive mutant SeVs were introduced into the M (G69E, T116A, and A183S) and HN (A262T, G264R, and K461G) proteins of SeV/⌬F to generate SeV/M ts HN ts ⌬F. The use of these mutations allows vector production at low temperature (32°C) and therapeutic use at body temperature (37°C) with diminished NTVLP formation. As expected, the formation of NTVLP by SeV/M ts HN ts ⌬F at 37°C was decreased to about 1/10 of that by SeV/⌬F, whereas the suppression of NTVLP formation did not cause either Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and is a member of the family Paramyxoviridae. The SeV genome contains six major genes arranged in tandem. The three virus-derived proteins nucleoprotein (NP), phosphoprotein (P), and large protein (L) form a ribonucleoprotein complex (RNP) with the SeV genomic RNA, and the RNP acts as a template for transcription and replication. Matrix protein (M) is a lining protein of the viral particle and is involved in the assembly of the particle. Two spike proteins, hemagglutinin-neuraminidase (HN) and fusion protein (F), mediate the attachment of virions and penetration of RNPs into infected cells, respectively (18). SeV replication appears to be independent of nuclear functions, and SeV does not have a DNA phase throughout its life cycle, so the virus is unable to transform cells by integrating its genetic information into the cellular genome (18).Since SeV infects and replicates in most mammalian cells, including human cells, and directs high-level expression of the genes it carries, vectors derived from SeV are expected to be useful for gene therapy by expressing therapeutic genes and vaccine antigens (16,21,29,36,38). In particular, we plan the clinical application of an SeV vector carrying human fibroblast growth factor 2 for the treatment of peripheral arterial disease. F-deficient SeV (SeV/⌬F) has thus been produced and demonstrated to be non-self-transmissible due to loss of the F gene in its genomic RNA (19). Recently, we have found that nontransmissible virus-like particles (NTVLP) are formed in cells infected with SeV/⌬F. The NTVLP formation should be reduced for the next-generation SeV vector rather than retained, although so far it has not been proven that NTVLP formation brings about any kind of adverse effect in vivo. In this study, we first characterized the virion formation of SeV/⌬F and showed that loss of the F gene from the SeV genome caused temperature sensitivity of NTVLP formation....

Dorsal vessel morphogenesis in Drosophila melanogaster serves as a superb system with which to study the cellular and genetic bases of heart tube formation. We used a cardioblast-expressed Toll-GFP transgene to screen for additional genes involved in heart development and identified tailup as a locus essential for normal dorsal vessel formation. tailup, related to vertebrate islet1, encodes a LIM homeodomain transcription factor expressed in all cardioblasts and pericardial cells of the heart tube as well as in associated lymph gland hematopoietic organs and alary muscles that attach the dorsal vessel to the epidermis. A transcriptional enhancer regulating expression in these four cell types was identified and used as a tailup-GFP transgene with additional markers to characterize dorsal vessel defects resulting from gene mutations. Two reproducible phenotypes were observed in mutant embryos: hypoplastic heart tubes with misaligned cardioblasts and the absence of most lymph gland and pericardial cells. Conversely, a significant expansion of the lymph glands and abnormal morphology of the heart were observed when tailup was overexpressed in the mesoderm. Tailup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand basic helix-loophelix transcription factor gene, with one site proven to be essential for the lymph gland, pericardial cell, and Svp/Doc cardioblast expression of Hand. Together, these results establish Tailup as being a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation and place this regulator directly upstream of Hand in these developmental processes.