Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays

Abstract

Embryonic development in Drosophila is characterized by an early phase during which a cellular blastoderm is formed and gastrulation takes place, and by a later postgastrulation phase in which key morphogenetic processes such as segmentation and organogenesis occur. We have focused on this later phase in embryogenesis with the goal of obtaining a comprehensive analysis of the zygotic gene expression that occurs during development under normal and altered environmental conditions. For this, a functional genomic approach to embryogenesis has been developed that uses highdensity oligonucleotide arrays for large-scale detection and quantification of gene expression. These oligonucleotide arrays were used for quantitative transcript imaging of embryonically expressed genes under standard conditions and in response to heat shock. In embryos raised under standard conditions, transcripts were detected for 37% of the 1,519 identified genes represented on the arrays, and highly reproducible quantification of gene expression was achieved in all cases. Analysis of differential gene expression after heat shock revealed substantial expression level changes for known heat-shock genes and identified numerous heat shock-inducible genes. These results demonstrate that highdensity oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large scale gene expression in Drosophila embryos. R ecently the genome of the first multicellular eukaryote Caenorhabditis elegans was completely elucidated (1). Sequencing of the Drosophila melanogaster genome has now also been carried out, and currently the corresponding putative open reading frames are being defined and verified (2). On the basis of this complete genomic information, it will now be important to determine the complex expression of all encoded genes and to analyze physiological as well as pathological phenomena from a global genetic perspective. Large-scale transcript analysis is made possible by DNA micro-or oligonucleotide arrays (3, 4), both of which allow the simultaneous monitoring of hundreds of mRNA expression profiles (5, 6). In this study, we used Drosophila high-density oligonucleotide arrays to monitor the simultaneous expression of zygotically active genes during the later postgastrulation stages of embryonic development (7-9). We analyzed the relative abundance levels of hundreds of embryonically expressed genes under normal physiological conditions and in response to heat shock (10). In embryos raised under normal conditions, we obtained highly reproducible quantification for 563 expressed genes corresponding to different functional classes. After a 36°C heat shock, we detected increases in expression levels for known heat-shock genes and identified numerous heat-shock-inducible genes. Materials and MethodsEmbryos. D. melanogaster Oregon R stocks were kept on standard cornmeal͞yeast͞agar medium at 25°C. Embryos were collected overnight on grape-juice plates for 12 h and were kept for a further 5 h at 25°C before RNA isolation...

Heat stress survival in the pre-adult stage of the life cycle in an intercontinental set of recombinant inbred lines ofDrosophila melanogaster

“…Some shsps were found to show clinal variation with latitude in D. melanogaster on the east coast of Australia (Frydenberg et al, 2003). Other heat-shock genes included within the above-mentioned QTL range (66D10-73B7) are hsp67Ba, hsp67Bb and hsp67Bc, with hsp67Bc being heat upregulated in a previous study in larvae (Leemans et al, 2000). Hsp83 is another heat-shock protein that was also heat upregulated both in the preadult stage of the life cycle (Leemans et al, 2000) and in adult flies (Sørensen et al, 2005).…”

“…Other heat-shock genes included within the above-mentioned QTL range (66D10-73B7) are hsp67Ba, hsp67Bb and hsp67Bc, with hsp67Bc being heat upregulated in a previous study in larvae (Leemans et al, 2000). Hsp83 is another heat-shock protein that was also heat upregulated both in the preadult stage of the life cycle (Leemans et al, 2000) and in adult flies (Sørensen et al, 2005). hsp83 maps within a QTL detected on the left arm of chromosome 3 in NT males.…”

“…Many candidate genes map within these QTL ranges (supplementary material TableS3). All of these candidate genes were either heat upregulated (Leemans et al, 2000;Ekengren and Hultmark, 2001;Sørensen et al, 2005) or differed in their respective expression levels between heat-resistant and heat-sensitive lines in D. melanogaster (McKechnie et al, 1998;Nielsen et al, 2006;Sørensen et al, 2007;Norry et al, 2009). Some of these candidate loci are involved in metabolic (e.g.…”

Quantitative trait loci for thermotolerance phenotypes in Drosophila melanogaster

“…The heat QTL H2 embraces a genomic region containing numerous positional candidate genes involved in stress resistance and response to heat. These include methuselah (mth; 61C), which has been shown to extend lifespan and increase stress resistance in mutant lines (Lin et al, 1998), and Hsp83 (63B) and DnaJ-1 (64E), both of which are upregulated in response to heat stress (Leemans et al, 2000). Thus, the tip of the left arm of chromosome 3 represents a genomic region with important effects on general heat-stress resistance.…”

Latitudinal and cold-tolerance variation associate with DNA repeat-number variation in the hsr-omega RNA gene of Drosophila melanogaster

“…This may be because different measures of heat tolerance have been used (for example Morgan and Mackay, 2006) or because relevant microarray studies have used arrays on which hsr-omega is not represented (Leemans et al, 2000;S rensen et al, 2005). Our central population family study gave no indication of an association of hsr-omega repeat variation with knockdown heat resistance.…”