Johan Staaf: Production of DNA microarrays for gene expression analysis.

Abstract
Gene expression profiling using microarrays in conjunction with the availability of complete genomic data for an increasing number of organisms has introduced a new paradigm in biomedical research. These achievements revolutionize biology, and offer a new means for solving problems within the life sciences. Array technology builds on classical hybridization chemistry. The novelty lies in the scale: currently, tens of thousands of gene fragments can be immobilized on single arrays, which allows the researcher a truly global view of the transcriptome in a single experiment

The biological complexity can be resolved by refining sample preparation. Instead of analyzing whole tissue biopsies, individual cell types can be isolated by laser capture micro-dissection, by fluorescence-activated cell sorting (FACS) or antibody coated magnetic beads, allowing only those cells showing a specific morphology or phenotype to be studied. This is a major advance, which is likely to rapidly become a required feature of advanced studies probing into the mechanistic analysis of cell and tissue differentiation. This increases the demands on sensitivity of the analytical methods, as well as on amplification methods to allow array technology to be applied starting from minimal amounts of RNA.

The Swegene DNA microarray resource center.
The technique had earlier been transferred from the National Human Genome Research Center at NIH to Dept Oncology and was moved and allowed to expand in new laboratory space at the BioMedical Center in conjunction to the Stem Cell Center in Lund, and is now up running in 2003. The aim of the centre is to provide south-west Swedish research groups access to state-of-the-art DNA microarray technology. This has been accomplished by establishing platforms and expertise for:

- Construction of high quality, high density microarray chips using different clone platforms.

- Design and construction of large scale PCR clone libraries through extensive automatisation involving liquid handling robots.

- Comprehensive RNA expression analysis in the most commonly used experimental systems (i.e. human, mouse and rat and eventually in fruit fly, zebra-fish, yeast and other species),

- High-density BAC arrays for comparative genomic hybridisation analysis of DNA copy gains and losses.

- Preparation of small sample amounts, RNA amplification (about to begin), labelling and
hybridisation.

- database handling and statistical analysis in cooperation with the bioinformatics center.

Recommended Reading:
Schulze A. , Downward J. Navigating gene expression using microarrays - a technology review. Nature Cell Biology, vol 3, August 2001.

Ewa Sitnicka: Receptor ligand interactions in the regulation of hematopoietic stem cell fate. Introduction by Sten Eirik Jacobsen

All blood cell lineages arise from common hematopoietic stem cells (HSC), which due to their capacity to self-renew maintain hematopoiesis throughout the lifespan of an individual. The enormous proliferative and differentiation potential of HSC can be illustrated by 3-5 million mature blood cells produced in man per second. The molecular mechanism regulating HSC fate decisions between self-renewal and lineage commitment remain poorly understood. However, evidence supports that blood lineage maturation and HSC self-renewal are regulated in part by hematopoietic cytokines interacting with their specific surface receptors.
Flt3 (or flk2) is a cytokine tyrosine kinase receptor primarily expressed at very early stages of hematopoiesis. Mice deficient in flt3 or flt3 ligand (FL) expression show deficient early B lymphopoiesis, but myeloid development is not affected. Although flt3 was cloned based on its expression in the HSC compartment, we demonstrated recently, that HSC in mouse bone marrow lack detectable cell surface flt3 expression (Adolfsson et al, Immunity 2001). To further address the role of flt3 and its ligand in early hematopoietic development we investigated hematopoiesis in FL-deficient mice, focusing the studies on the role of FL in regulation of HSC, common lymphoid progenitors (CLP) and common myeloid progenitors (CMP). Whereas HSC and common myeloid progenitors are unaffected, FL-deficient mice show striking reductions in CLP, suggesting a role for FL in early lymphoid development throughout the generation of CLP form HSC (Sitnicka et al, Immunity 2002). An indispensable role of flt3 signaling in B cell development was established through studies in mice double deficient in FL and Interleukin-7 (IL-7) receptor expression. Although both of these cytokines have been demonstrated to be important regulators of B cell development, normal levels of immunoglobulins are produced in mice deficient in FL or IL-7R expression. However, based on potent synergistic interactions between these two ligands in vitro we hypothesized that the concerted action of FL; acting at very early stages of lymphopoiesis and the lymphoid lineage specific factor IL-7 might be indispensable for B cell development in vivo. This hypothesis is supported by the finding that mice double deficient in FL and IL-7 receptor expression completely lack mature conventional IgM+ B cells, IgA+ plasma cells, B1 B cells and fail to produce immunoglobulins.

Recommended literature:
1. Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem cells, cancer, and cancer stem cells. Nature 414, 105-11. (2001).
2. Lyman, S. D. & Jacobsen, S. E. c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood 91, 1101-34. (1998).
3. Fry, T. J. & Mackall, C. L. Interleukin-7: from bench to clinic. Blood 99, 3892-904. (2002).
4. Adolfsson, J. et al. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c- kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15, 659-69. (2001).
5. Sitnicka, E. et al. Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity 17, 463-72. (2002).
6. Veiby, O. P., Lyman, S. D. & Jacobsen, S. E. Combined signaling through interleukin-7 receptors and flt3 but not c- kit potently and selectively promotes B-cell commitment and differentiation from uncommitted murine bone marrow progenitor cells. Blood 88, 1256-65. (1996).

Florian Lueders: Slalom encodes a PAPS transporter essential for segment polarity and dorsal ventral axis determination in drosophilia. Introduction by Udo Haecker

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate. Here we report the first molecular identification of a gene that encodes a transmembrane protein required to transport adenosine 3'-phosphate 5'-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which we call slalom, display defects in Wg and Hh signaling, which are likely due to the lack of sulfation of glycosaminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal-ventral axis determination suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal-ventral determinant pipe.

Keywords: Hedgehog/Glycosaminoglycan/Pipe/Sulfation/Wingless

Introduction
Secreted signaling molecules of the FGF, Hh, TGFb and WNT families rely on proteoglycans (PGs) for efficient activation of their respective signaling pathways (reviewed in Perrimon and Bernfield, 2000). PGs consist of secreted or transmembrane core proteins to which glycosaminoglycan (GAG) side chains are attached at specific consensus sites. In Drosophila, the secreted PG Perlecan (Park et al., 2003), the transmembrane PG Syndecan (Spring et al., 1994), and two members of the Glypican-family of glycosylphosphatidylinositol (GPI)-anchored PGs have been identified. Phenotypes associated with loss of function mutations of the Glypican-encoding genes dally (Nakato et al., 1995) and dally-like (dlp) (Baeg et al., 2001; Khare and Baumgartner, 2000) have revealed the requirement of these PGs for efficient activation of several signal transduction pathways.
The function of PGs is critically dependent on the integrity of the attached GAGs. GAGs are unbranched polysaccharide chains, which are synthesized on proteoglycan core proteins in the Golgi and undergo complex modification reactions before the PG, that they are attached to, is transported to the cell surface. In the case of Glypican, heparan sulfate (HS) chains, which consist of a sugar backbone of alternating units of N-acetyl-glucosamine (GlcNAc) and glucuronic acid (GlcA) are synthesized on the core protein. The nucleotide sugar substrates for this reaction are synthesized in the cytoplasm and must be transported into the Golgi. In Drosophila, the activated precursor UDP-GlcA is synthesized from UDP-glucose by the enzymatic activity of the sugarless (sgl) (Binari et al., 1997; Häcker et al., 1997; Haerry et al., 1997) gene product, a homolog of mammalian UDP-glucose dehydrogenases. The gene product of fringe connection (frc), a predicted ER/Golgi multi-pass transmembrane protein has been shown to transport UDP-GlcA and UDP-GlcNAc from the cytosol into the Golgi (Goto et al., 2001; Selva et al., 2001). Mutations in either gene severely affect the Wg and FGF signaling pathways. Elongation of the HS chains requires the activity of HS polymerases. tout-velu (ttv), encodes a protein with homology to the mammalian HS co-polymerase EXT1and has been demonstrated to be required specifically for Hh signaling (Bellaiche et al., 1998). Subsequent to their synthesis, GAGs undergo multiple modifications such as epimerization and sulfation. Mutations in sulfateless (sfl), a homologue of vertebrate N-deacetylase/ N-sulfotransferases (NDST) lead to a severe reduction in the activity of the Wg, Hh and FGF signaling pathways (Lin et al., 1999; Lin and Perrimon, 1999). A characteristic feature of all mutations in genes involved in GAG biosynthesis is, that their segment polarity phenotypes can be rescued by ectopic expression of wg or hh, suggesting that GAGs are not essential components of the respective signaling cascades but accessory factors most likely required for the proper distribution of extracellular signaling molecules throughout morphogenetically active tissues in vivo.

GAGs have also been proposed to play a role in the determination of the dorsal-ventral (D/V) axis of the Drosophila embryo (Sen et al., 1998). The D/V polarity of the embryo is established during oogenesis by asymmetric expression of the key D/V determinant pipe (pip) in the follicle cell epithelium. pip expression in the ventral follicle cell layer is necessary and sufficient to trigger a serine-protease cascade in the perivitelline space, which leads to the generation of an active ligand for the transmembrane receptor Toll (Tl). Activation of Tl on the ventral side of the embryo results in a gradient of nuclear localization of the transcription factor Dorsal, which patterns the D/V axis (reviewed in Amiri and Stein, 2002). Based on sequence similarity to a family of vertebrate enzymes, and its localization in the Golgi apparatus, pip has been hypothesized to encode a heparansulfate 2-O-sulfotransferase (HSST). However, neither the enzymatic activity nor the substrate specificity of pip have been demonstrated directly.

Sulfation of secreted molecules occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to be present within that organelle. In Drosophila, PAPS is synthesized in the cytoplasm by PAPS-synthetase (Jullien et al., 1997), which incorporates both ATP-sulfurylase and Adenosine 5'-phosphosulfate-kinase (APS-kinase) activity. PAPS must be transported into the Golgi thereafter (Lyle et al., 1994) to serve as a substrate for sulfotransferases. Here, we report the first molecular identification and functional characterization of a PAPS-transporter. Mutations in this gene, which we call slalom (sll), are associated with defects in multiple signaling pathways, including Wg and Hh signaling. A phenotypic analysis suggests that the effects of sll on signal transduction are caused by its requirement for GAG modification. We present evidence that sll is also required to supply PAPS to the machinery initiating the establishment of embryonic D/V polarity, supporting the view that Pipe protein is a sulfotransferase.