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 Polycomb 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.

Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia inDrosophila. 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 Drosophiladevelopment 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.

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.

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.