We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

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.

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.

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....

A new recombinant Sendai virus vector (SeV/⌬M), in which the gene encoding matrix (M) protein was deleted, was recovered from cDNA and propagated in a packaging cell line expressing M protein by using a Cre/loxP induction system. The titer of SeV/⌬M carrying the enhanced green fluorescent protein gene in place of the M gene was 7 ؋ 10 7 cell infectious units/ml or more. The new vector showed high levels of infectivity and gene expression, similar to those of wild-type SeV vector, in vitro and in vivo. Virus maturation into a particle was almost completely abolished in cells infected with SeV/⌬M. Instead, SeV/⌬M infection brought about a significant increase of syncytium formation under conditions in which the fusion protein was proteolytically cleaved and activated by trypsin-like protease. This shows that SeV/⌬M spreads markedly to neighboring cells in a cell-to-cell manner, because both hemagglutinin-neuraminidase and active fusion proteins are present at very high levels on the surface of cells infected with SeV/⌬M. Thus, SeV/⌬M is a novel type of vector with the characteristic features of loss of virus particle formation and gain of cell-to-cell spreading via a mechanism dependent on the activation of the fusion protein.

The core promoter is a critical DNA element required for accurate transcription and regulation of transcription. Several core promoter elements have been previously identified in eukaryotes, but those cannot account for transcription from most RNA polymerase II-transcribed genes. Additional, as-yet-unidentified core promoter elements must be present in eukaryotic genomes. From extensive analyses of the hepatitis B virus X gene promoter, here we identify a new core promoter element, XCPE1 (the X gene core promoter element 1), that drives RNA polymerase II transcription. XCPE1 is located between nucleotides ؊8 and ؉2 relative to the transcriptional start site (؉1) and has a consensus sequence of G/A/T-G/C-G-T/C-G-G-G/A-A-G/C ؉1 -A/C. XCPE1 shows fairly weak transcriptional activity alone but exerts significant, specific promoter activity when accompanied by activator-binding sites. XCPE1 is also found in the core promoter regions of about 1% of human genes, particularly in poorly characterized TATA-less genes. Our in vitro transcription studies suggest that the XCPE1-driven transcription can be highly active in the absence of TFIID because it can utilize either free TBP or the complete TFIID complex. Our findings suggest the possibility of the existence of a TAF1 (TFIID)-independent transcriptional initiation mechanism that may be used by a category of TATA-less promoters in higher eukaryotes.Transcriptional initiation is a key regulatory step of gene expression. Regulation of transcriptional initiation is carried out by a complex network of interactions between cis-acting DNA elements and DNA-binding transcription factors and also among many transcription factors, including sequencespecific DNA-binding transcriptional regulators, coregulators, and general (basal) transcriptional machinery (i.e., general transcription factors [GTFs] and RNA polymerase [pol]) (22, 26, 33-35, 41, 51, 54).The DNA regions that specify the transcriptional program of each gene contain two functionally distinct regions, the core promoter (for reviews, see references 9, 61, and 62) and the regulatory regions (e.g., enhancers, silencers, etc.). The core promoter is the minimum essential region necessary for accurate transcription. The core promoter typically comprises about 40 nucleotides (nt) and contains functional subregions called core promoter elements. When transcription starts, the core promoter elements are recognized by some of the GTFs or by other factors that trigger the assembly of a large protein complex that consists of GTFs and RNA polymerase (i.e., the transcriptional preinitiation complex [PIC]) at the core promoter. The transcriptional initiation complex positions RNA polymerase at the correct site and makes it possible to start RNA synthesis accurately. Thus, core promoter elements play an essential role in specifying transcription start sites. Core promoter elements also determine the specific transcriptional properties of each core promoter, dictating (i) which RNA polymerase among the class I, II, and III systems sh...

Bag of Marbles (Bam) is a stem cell differentiation factor in the Drosophila germ line. Here, we demonstrate that Bam has a crucial function in the lymph gland, the tissue that orchestrates the second phase of Drosophila hematopoiesis. In bam mutant larvae, depletion of hematopoietic progenitors is observed, coupled with prodigious production of differentiated hemocytes. Conversely, forced expression of Bam in the lymph gland results in expansion of prohemocytes and substantial reduction of differentiated blood cells. These findings identify Bam as a regulatory protein that promotes blood cell precursor maintenance and prevents hemocyte differentiation during larval hematopoiesis. Cell-specific knockdown of bam function via RNAi expression revealed that Bam activity is required cell-autonomously in hematopoietic progenitors for their maintenance. microRNA-7 (mir-7) mutant lymph glands present with phenotypes identical to those seen in bam-null animals and mutants double-heterozygous for bam and mir-7 reveal that the two cooperate to maintain the hematopoietic progenitor population. By contrast, analysis of yan mutant lymph glands revealed that this transcriptional regulator promotes blood cell differentiation and the loss of prohemocyte maintenance. Expression of Bam or mir-7 in hematopoietic progenitors leads to a reduction of Yan protein. Together, these results demonstrate that Bam and mir-7 antagonize the differentiation-promoting function of Yan to maintain the stem-like hematopoietic progenitor state during hematopoiesis.