Cell division and subsequent programmed cell death in imaginal discs of Drosophila larvae determine the final size of organs and structures of the adult fly. We show here that nitric oxide (NO) is involved in controlling the size of body structures during Drosophila development. We have found that NO synthase (NOS) is expressed at high levels in developing imaginal discs. Inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, whereas ectopic expression of NOS in larvae has the opposite effect. Blocking apoptosis in eye imaginal discs unmasks surplus cell proliferation and results in an increase in the number of ommatidia and component cells of individual ommatidia. These results argue that NO acts as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.

The activity of the Drosophila gene trithorax is required to maintain the proper spatial pattern of expression of multiple homeotic genes of the Bithorax and Antennapedia complexes, trithorax encodes two large protein isoforms of >400 kD. We have detected its products at 16 discrete sites on larval salivary gland polytene chromosomes, 12 of which colocalize with binding sites of several Poly comb group proteins. The intensity of trithorax protein binding is strongly decreased in larvae carrying mutations in another trithorax group gene ash-1, and in the Polycomb group gene pco/E(z). A strong trithorax binding site was found at the cytological location of the fork head gene, a region-specific homeotic gene not located within a homeotic complex. Further analysis showed that trithorax protein binds at ectopic sites carrying fork head sequences in transformed lines. Trithorax binding occurs within an 8.4-kb regulatory region that directs fork head expression in several embryonic tissues including salivary glands. Consistently, expression of endogenous fork head RNA is greatly reduced in trithorax mutant embryos and in larval tissues. These results show that trithorax maintains expression of target genes by interaction with their regulatory regions and that this interaction depends on the presence of at least some of the other trithorax and Polycomb group proteins.[Key Words: Drosophila-, trithorax-, chromosomal binding; regulation of expression; fork head]

Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. We cloned and analyzed the only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS). It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. We identified eight new transcripts that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene lead to premature termination of the open reading frame. As a result, none of the alternative transcripts encode an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, we demonstrate that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. Our results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene. Nitric oxide (NO)1 is involved in vasodilation, neurotransmission, and immunity in mammals (see Ref. 1 for review). It is produced by a family of nitric-oxide synthases (NOS), which are encoded by three distinct genes in the mammalian genome. These genes are expressed throughout development as well as in the adult animal. Their transcriptional regulation is highly complex; a range of alternative promoters, splice sites, and polyadenylation signals is used to generate families of transcripts and proteins from each chromosomal gene (see Refs. 2-4 for review).In Drosophila NO has been shown to be involved in imaginal disc development, synaptogenesis, formation of retinal projection pattern, response to hypoxia, and behavioral responses (see Ref. 5 for review). The gene for Drosophila NO synthase, dNOS, is located on the second chromosome at cytological position 32B (6). The major product of the gene, dNOS1, encodes a protein that bears a strong resemblance to all three NOS isoforms of mammals, with the highest homology to neuronal NOS (nNOS) (6). Several attempts to clone putative orthologs of mammalian endothelial or inducible forms of NOS (eNOS or iNOS) from various Drosophila cDNA or genomic libraries have not revealed any other NOS loci in the fruit fly's genome. 2 The recently released complete sequence of the Drosophila genome (7) has further confirmed our conclusion that the dNOS locus represents the only gene for NO synthase in Drosophila.To explore the role of NO in fruit fly physiology and development, we sought to determine the structure of the Drosophila NOS gene to search for alternative transcripts and proteins and...

Mechanisms controlling the transition of precursor cells from proliferation to differentiation during organism development determine the distinct anatomical features of tissues and organs. NO may mediate such a transition since it can suppress DNA synthesis and cell proliferation. Inhibition of NOS activity in the imaginal discs of Drosophila larvae results in hypertrophy of tissues and organs of the adult fly, whereas ectopic overexpression of NOS has the reciprocal, hypotrophic, effect. Furthermore, NO production is crucial for the establishment of ordered neuronal connections in the visual system of the fly, indicating that NO affects the acquisition of the differentiated phenotype by the neural tissue. Increasing evidence points to a broad role that NO may play in animal development by acting as an essential negative regulator of precursor cell proliferation during tissue and organ morphogenesis.Keywords: nitric oxide; drosophila; differentiation; synaptogenesis; organ development Abbreviations: APF, after puparium formation; BrdU, 5-bromodeoxyuridine; cGMP, 3'5'cyclic guanosine monophosphate; DPH, nicotinamide adenine dinucleotide phosphate; OS, nitric oxide synthase; Rb, retinoblastoma Organ and tissue development require a tightly controlled program of cell proliferation followed by growth arrest and differentiation and, often, programmed cell death. The balance between the number of cell divisions and the extent of subsequent programmed cell death determines the final size of a tissue or an organ (reviewed in 1 ± 3 ). Although much of the cellular machinery that controls cell division per se is well understood (reviewed in 4 ± 8 ) less is known about the signals that cause discrete groups of cells within organs to stop dividing upon reaching an appropriate cell number. These signals should relate cell cycle progression to information about the size of a domain of adjacent cells, and they probably involve some as yet undetermined inter-and intracellular second messenger molecules. These messengers might include multifunctional signaling molecules that would couple together several events, such as cessation of cell cycle progression, implementation of programmed cell death, and acquisition of specific traits that define a differentiated tissue.Nitric oxide (NO) is a versatile diffusible second messenger implicated in numerous physiological functions in mammals, ranging from dilation of blood vessels and muscle relaxation to immune responses and potentiation of synaptic transmission (reviewed in 9 ± 12 ). It has also been shown to affect gene expression at the level of transcription 13 ± 16 or translation. 17 ± 19 NO is produced by nitric oxide synthase (NOS) in almost all cell types. Various isoforms of NOS are expressed throughout animal development, marking characteristic steps in tissue differentiation. 20 Recent evidence indicates that NO may be an important player in the program of development, directing the transition out of the proliferating state to differentiation and affecting the acquis...

Hematopoietic stem cells give rise to various multipotent progenitor populations, which expand in response to cytokines and which ultimately generate all of the elements of the blood. Here we show that it is possible to increase the number of stem and progenitor cells in the bone marrow (BM) by suppressing the activity of NO synthases (NOS). Exposure of mice to NOS inhibitors, either directly or after irradiation and BM transplantation, increases the number of stem cells in the BM. In the transplantation model, this increase is followed by a transient increase in the number of neutrophils in the peripheral blood. Thus, our results indicate that NO is important for the control of hematopoietic stem cells in the BM. They further suggest that suppression of NO synthase activity may allow expansion of the number of hematopoietic stem and progenitor cells or neutrophils for therapeutic purposes.