DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication     

Temporal and spatial expression of the four Igf ligands and two Igf type 1 receptors in zebrafish during early embryonic development

The insulin-like growth factor (Igf) family is an evolutionarily conserved system essential for normal growth and development in vertebrates. Unlike mammals, four distinct Igf ligands (Igf1, Igf2a, Igf2b and Igf3) and two Igf type 1 receptors (Igf1ra and Igf1rb) are present in zebrafish. However, the localization of these multiple ligands and receptors especially the recently discovered igf3 during early development of zebrafish is poorly understood. In this study, detailed expression patterns of these components of the Igf system during embryogenesis of zebrafish were analyzed. It was found that igf1 is specifically expressed in the trigeminal ganglia region from 18 hpf to 72 hpf, while igf2a is restricted to the caudal regions of the notochord from 14 hpf to 18 hpf as well as in the midbrain, dorsal hind brain and otic vesicle at 24 hpf. On the other hand, igf2a is highly expressed in the midbrain and pharyngeal arch region at 48 hpf, followed by its appearance in the liver and brain at 72 hpf, while igf2b is restricted to the floor plate and hypochord from 12 hpf to 18 hpf, and strong expression is also detected in the midbrain and dorsal hind brain at 24 hpf. The teleost specific igf3 is highly expressed in the pharyngeal arch region before 24 hpf, but is then restricted to the sternohyoideus after 48 hpf. The receptor subtype igf1ra is ubiquitously expressed before 24 hpf but is confined to the brain at 72 hpf. However, igf1rb is widely expressed before 10 hpf, but is more confined to the brain region at 24 hpf and 72 hpf. This dynamic temporal-spatial expression during embryogenesis of zebrafish, together with the unique and overlapping expression patterns of the Igf ligands and receptors suggest the coordination of the divergent functions of the Igf system during early development in zebrafish.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

Gene expression profiling of mouse postnatal cerebellar development using oligonucleotide microarrays designed to detect differences in glycoconjugate expression

Differences in gene expression patterns between adult and postnatal day 7 (P7) mouse cerebellum, at the peak of granule neuron migration, were analyzed by hybridization to the GLYCOv2 glycogene array. This custom designed oligonucleotide array focuses on glycosyl transferases, carbohydrate-binding proteins, proteoglycans and related genes, and 173 genes were identified as being differentially expressed with statistical confidence. Expression levels for 11 of these genes were compared by RT-PCR, and their differential expression between P7 and adult cerebellum confirmed. Within the group of genes showing differential expression, the sialyltransferases (SiaTs) and GalNAc-Ts that were elevated at P7 prefer glycoprotein substrates, whilst the SiaTs and GalNAc-Ts that were elevated in the adult preferentially modify glycolipids, consistent with a role for gangliosides in maintaining neuronal function in the adult. Also within this group, three proteoglycans--versican, bamacan and glypican-2--were elevated at P7, along with growth factor midkine, which is known to bind to multiple types of proteoglycans, and fibroblast growth factor receptor 1, whose activity is known to be influenced by heparan sulfate proteoglycans. Two sulfotransferases that can modify the extent of proteoglycan sulfation were also differentially regulated, and may modify the interaction of a subset of proteoglycans with their binding partners during cerebellar development. Bamacan, glypican-2 and midkine were shown to be expressed in different cell types, and their roles in cerebellar development during granule neuron migration and maturation are discussed.     

Microarray gene expression analysis free of reverse transcription and dye labeling

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

Expression analysis of nel during zebrafish embryonic development

Nel is a multimeric extracellular glycoprotein which predominantly expressed in the nervous system and play an important role in neural development and functions. There are three nel paralogues included nell2a, nell2b, and nell3 in zebrafish, while systematic expression analysis of the nel family is still lacking. In this study, we performed a phylogenetic analysis on 7 species, in different species the nell2a are highly conserved, as is nell2b. Then, the expression profiles of nell2a, nell2b and nell3 were detected by in situ hybridization in zebrafish embryo, and the result showed that nel genes highly enriched in the central nervous system, but distributed in different regions of the brain. In addition, nell2a is also expressed in the olfactory pit, spinal cord, otic vesicle and retina (ganglion cell layer), nell2b was detected to express in gill arches, olfactory epithelium, olfactory pit, spinal cord, photoreceptor and retina (ganglion cell layer), it should be noted that the expression of nell3 is special, was only detected at 96 hpf in the brain and spinal cord of zebrafish. Overall, our results indicate that nell2a and nell2b genes are expressed in the nervous system and eyes of zebrafish embryo, while nell3 is expressed in different regions in the nervous system. The phylogenetic analysis also shows that nell3 sequences are significantly different from nell2a and nell2b. This study provides new evidence to better understand the role of nel in zebrafish embryo development.     

Microarray analysis of peroxisome proliferator-activated receptor-gamma induced changes in gene expression in macrophages

We used a combination of expression microarray and Northern blot analyses to identify target genes for peroxisome proliferator-activated receptor (PPAR) gamma in RAW264.7 macrophages. PPARgamma natural ligand 15-deoxy-Delta(12,14) prostaglandin and synthetic ligands ciglitazone and rosiglitazone increased the expression of scavenger receptor CD36 and ATP-binding cassette transporter A1, as well as adipophilin (a lipid droplet coating protein involved in intracellular lipid storage and transport), calpain (a protease implicated in ABCA1 protein degradation), and ADAM8 (a disintegrin and metalloprotease protein involved in cell adhesion). These findings are relevant to understanding the effect of PPARgamma activation on gene expression and cognate pathways in macrophages.     

Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis

T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish, Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development.