Overview of the in situ production pipeline. (a-e) Examples illustrating each step of the high-throughput production of RNA in situ patterns. (a) Photograph of an agarose gel showing PCR products from a single 96-well plate. The vertical lines mark failed PCR reactions. (b) Nylon membrane spotted with 96 RNA in situ probes and stained to reveal the incorporation of digoxigenin. Missing or weak spots indicate a failed probe reaction. (c) Four wells of a 96-well plate, each containing about 200 embryos. (d) Low-resolution photograph of stained embryos. (e) High-resolution photograph of an individual embryo. All image and textual data are entered into a MySQL relational database and integrated with microarray data and previously published expression patterns.

Imaging expression patterns during embryogenesis. Screen shot of the web-based tool used to organize and annotate captured images. The figure shows the categorization of images in six stage ranges, reflecting developmental time, presented left to right. The annotation terms are similarly arranged from left to right and grouped from top to bottom according to related organ systems. Groups of images at a given stage range will be associated with groups of annotation terms appropriate for that stage range. Microarray profiles and links to public databases are also included.

Visualization of dynamic developmental processes. (a-e) Series of five embryos stained with a probe (bgm) highlighting the fat-body primordium and revealing dynamic aspects of segmental fat-body specification. Ventral view with anterior to the left. (f-j) Series of five embryos stained with a probe (CG4829) visualizing the lamellocyte precursors. The staining reveals the spreading pattern of lamellocytes across the embryo and the 'hitchhiking' of migrating cells on the retracting germ band.

Comparison of image and microarray data for gene CG4702. (a) A microarray expression profile of the gene CG4702, represented by EST clone LD43816. Consecutive time points representing RNA samples from differentially aged 1-h embryo collections are presented on the x-axis, while the y-axis represents the scale of absolute expression indexes as analyzed by dChip analysis software [37]. Error bars represent standard error of the measurements, which were carried out independently on three separate embryo collections; the number next to each bar is the mean of the three measurements. The color of the bar reflects the categorization of each measurement on the basis of a statistical analysis performed by the Affymetrix Microarray Suite software package. Green represents present; red, absent. Because of the sensitivity limitations of the GeneChip measurements and the conservative nature of the software, absent calls do not necessarily imply lack of gene expression. The secondary x-axis correlates the microarray sample time points with stages of embryogenesis. (b) Images of six staged embryos illustrating expression during development of the same gene characterized above by microarray analysis. Each image comes from a different stage range (stages 1-3, 4-6, 7-8, 9-10, 11-12, and 14-16). Lateral view, anterior to the left.

Correlation of image and microarray data. (a-n) Fourteen examples of correlations between microarray and image data for genes with known expression patterns. Microarray expression profiles are shown on the left. Red is absent, green is present, and vertical lines on bars represent error bars of triplicate measurements. On the right are six representative images, one for each stage range specified in Figure 4, ordered according to developmental time to allow visual correlation with the corresponding array profile. Anterior to the left. The most straightforward comparisons occur when gene expression comes on (e,n) or is turned off abruptly (a,b), corresponding to the absence or presence of in situ staining respectively. In many cases, the absent/present call misses the expression of genes confined to a small subset of tissues (c,m). (l) Gene-expression levels increase, followed by an increase in staining intensity that occupies approximately the same proportion of the embryo. (f) The increase in microarray intensity reflects an increase in the number of cells in the embryo showing gene expression across time. (c) Fading expression is indicative of the restriction of gene expression to a smaller subset of cells as development proceeds. Frequently, a microarray profile will show both types of fluctuations, and in that case the visual correlation is rather subjective (g,h), unless accompanied by a clear-cut qualitative change (d,e,i). (k) Genes transcribed both maternally and zygotically have no 'off' period during our developmental time course. (j) The decrease in abundance of maternal transcript often overlaps with emergence of zygotic transcript, leading to the flattening of the early portion of the microarray profile.

Textual annotations using controlled vocabularies. (a) General categories of anatomical terms and the developmental relationships between them. We distinguish four types of anatomical structures: 'anlage in statu nascendi', 'anlage', 'primordium' and differentiated 'organs'. Below each heading are examples of staining patterns that illustrate a specific instance of that category of developmental structure. The examples are not developmentally related. Anlage in statu nascendi, anlage and primordium become suffixes to the specific name of a structure, such as dorsal ectoderm anlage in statu nascendi, head mesoderm anlage and Malpighian tubule primordium. Differentiated organs are named without a suffix, for example fat body. (b) Embryos at the indicated stages stained with a probe for the gene single minded reveal the developmental origin of the midline glia. Ventral view, anterior to the left.

Screenshot of the result page of our publicly available search tool. The first four of the 14 genes returned by a query for genes expressed in the 'embryonic optic lobe' are shown. The 'Gene' column gives the gene name, gene identifier, EST identifier, cytological position and GO function assignments, where available. The gene name serves as a link to access the complete expression report page for that gene. Array profiles and images can be enlarged in a separate window by clicking on the thumbnail image. In the column labeled 'Body Part' is a list of all annotation terms that have been assigned to that gene, with the query subject (in this case, embryonic optic lobe) highlighted by bold italics. Each annotation term is hyperlinked to the ImaGO Gene Ontology browser.

Hierarchical clustering of controlled vocabulary annotations. Columns of the matrix represent the 99 annotation terms from the controlled vocabulary that correspond to differentiated organs. Rows of the matrix represent 1,257 genes that are expressed in at least one of those embryonic structures. A black matrix cell indicates that a gene is expressed in the given structure. Both rows and columns are clustered using a binary distance metric and hierarchical complete linkage clustering. Green shading highlights clusters of embryonic structures that are part of the nervous system, cyan shading highlights clusters formed by components of the muscle system. Genes expressed specifically in the embryonic fat body are highlighted by a red rectangle, two separate clusters of genes expressed in the tracheal system are surrounded by blue and magenta rectangles. A cluster of genes expressed in the peripheral nervous system and not in the central nervous system is enclosed by the yellow rectangle.

Examples of interesting observations from this project. (a-d) Three adjacent Brachyury/T-box genes - (a) CG5093, (b) CG5107, (c) Dorsocross - show remarkably similar expression patterns at cellular blastoderm and are located next to each other on the chromosome (d). (e-i) Expression patterns of five genes with homology to metabolic and detoxification enzymes: (e) hydroxyacylglutathione hydrolase, CG9026; (f) cytochrome P450, Cyp310a; (g) multi-drug resistance gene, Mdr49; (h) xanthine dehydrogenase, rosy; and (i) transketolase, CG8036. (j) Dorsal view of a stage-8 embryo stained with a probe that reveals apically localized mRNAs (CG18375) within the developing hindgut epithelium, contrasted with basally localized mRNA (CG11207) in the same cells (k). (l) Close-up of (j) highlighting the apically localized mRNA in the epithelium surrounding the invaginating hindgut pocket with pole cells (pc). A single epithelial cell is outlined. (m) Close-up of (K) revealing an equivalent region of an embryo as (l) and highlighting the basally localized mRNA. (n) Portion of the dorsal side of a pre-blastoderm embryo stained for mRNA (CG1962) that appears to be localized to an unidentified subnuclear compartment. (o) Close-up of five nuclei from (n).