eChickAtlas: an introduction to the database.

The precise control of gene expression is critical in embryonic development. Quantitative assays, such as microarrays and RNA sequencing, provide gene expression levels for a large number of genes, but do not contain spatial information. In contrast, in situ methods, such as in situ hybridization and immunohistochemistry, provide spatial resolution, but poor quantification and can only reveal the expression of one, or very few genes at a time. Furthermore, the usual methods of documenting the results, by photographing whole mounts or sections, makes it very difficult to assess the three-dimensional (3D) relationships between expressing and nonexpressing cells. Optical projection tomography (OPT) can capture the full 3D expression pattern in a whole embryo at a reasonable level of resolution and at moderately high throughput. A large database containing spatio-temporal patterns of expression for the mouse (e-Mouse Atlas Project, EMAP, www.emouseatlas.org) has been created, incorporating 3D information. Like the mouse, the chick is an important model in developmental biology and translational studies. To facilitate comparisons between these important model organisms, we have created a 3D anatomical atlas, accompanied by an anatomical ontology of the chick embryo and a database of gene expression patterns during chick development. This database is publicly available (www.echickatlas.org).

Comparative analysis of 3D expression patterns of transcription factor genes and digit fate maps in the developing chick wing.

Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.

ZebraFISH: fluorescent in situ hybridization protocol and three-dimensional imaging of gene expression patterns.

We present a method and protocol for fluorescent in situ hybridization (FISH) in zebrafish embryos to enable three-dimensional imaging of patterns of gene expression using confocal laser scanning microscopy. We describe the development of our protocol and the processing workflow of the three-dimensional images from the confocal microscope. We refer to this protocol as zebraFISH. FISH is based on the use of tyramide signal amplification (TSA), which results in highly sensitive and very localized fluorescent staining. The zebraFISH protocol was extensively tested and here we present a panel of five probes for genes expressed in different tissues or single cells. FISH in combination with confocal laser scanning microscopy provides an excellent tool to generate three-dimensional images of patterns of gene expression. We propose that such three-dimensional images are suitable for building a repository of gene expression patterns, complementary to our previously published three-dimensional anatomical atlas of zebrafish development (bio-imaging.liacs.nl/). Our methodology for image processing of three-dimensional confocal images allows an analytical approach to the definition of gene expression domains based on the three-dimensional anatomical atlas.

Gene expression and digit homology in the chicken embryo wing.

The bird wing is of special interest to students of homology and avian evolution. Fossil and developmental data give conflicting indications of digit homology if a pentadactyl "archetype" is assumed. Morphological signs of a vestigial digit I are seen in bird embryos, but no digit-like structure develops in wild-type embryos. To examine the developmental mechanisms of digit loss, we studied the expression of the high-mobility group box containing Sox9 gene, and bone morphogenetic protein receptor 1b (bmpR-1b)-markers for precondensation and prechondrogenic cells, respectively. We find an elongated domain of Sox9 expression, but no bmpR-1b expression, anterior to digit II. We interpret this as a digit I domain that reaches precondensation, but not condensation or precartilage stages. It develops late, when the tissue in which it is lodged is being remodeled. We consider these findings in the light of previous Hoxd-11 misexpression studies. Together, they suggest that there is a digit I vestige in the wing that can be rescued and undergo development if posterior patterning cues are enhanced. We observed Sox9 expression in the elusive "element X" that is sometimes stated to represent a sixth digit. Indeed, incongruity between digit domains and identities in theropods disappears if birds and other archosaurs are considered primitively polydactyl. Our study provides the first gene expression evidence for at least five digital domains in the chick wing. The failure of the first to develop may be plausibly linked to attenuation of posterior signals.