Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning

Abstract

In a large-scale gene expression screen 1765 randomly picked cDNAs were analyzed by whole-mount in situ hybridization in Xenopus embryos. Two hundred and seventy three unique, differentially expressed genes were identified, 204 of which are novel in Xenopus. Partial DNA sequences and expression patterns were documented and assembled into a database, 'AXelDB'. Approximately 30% of cDNAs analyzed represent differentially expressed genes and about 5% show highly regionalized expression. Novel marker genes and potential developmental regulators were found. Differential expression of mitochondrial genes was observed. Marker genes were used to study regionalization of the entire gastrula as well as the tail forming region and the epidermis of the tailbud embryo. Four 'synexpression' groups representing genes with shared, complex expression pattern that predict molecular pathways involved in patterning and differentiation were identified. According to their probable functional significance these groups are designated as Delta1, Bmp4, ER-import and Chromatin group. Within synexpression groups, a likely function of genes without sequence similarity can be predicted. The results indicate that synexpression groups have strong prognostic value. A cluster analysis was made by comparing gene expression patterns to derive a novel parameter, 'tissue relatedness'. In conclusion, this study describes a semi-functional approach to investigate genes expressed during early development and provides global insight into embryonic patterning.

Pilot morpholino screen in Xenopus tropicalis identifies a novel gene involved in head development

“…2B,g). We have not been able to detect expression in later stage embryos, but in X. laevis, there is some expression over the mid-hindbrain region (Gawantka et al, 1998). However, in MO-injected tadpole stage embryos, expression is detected in the anterior endoderm, in a stripe posterior to the eyes, and in the posterior ventral mesoderm (Fig.…”

“…Targeted genes are grouped according to a functional class. The expressed sequence tag (EST) name for each sequence as assigned by Gawantka et al, (1998) and http://www.dkfz-heidelberg.de/abt0135/axeldb.htm for Xenopus laevis is given next to the best protein match for each translated sequence. Where a protein may not represent an orthologue but is homologous, it is suffixed by "like."…”

“…The gene sequences are known a priori, and once an interesting phenotype has been identified, the expression pattern can be easily determined. In fact, the gene sequences and/or the expression patterns can be used to select for inactivation candidate genes of particular specifications, as we have done here for neurally expressed genes that were previously identified in an expression screen (Gawantka et al, 1998). Furthermore, because the X. tropicalis cDNAs are already cloned in an expression vector, the phenotype of overexpressing the gene(s) of interest can be determined without delay.…”

“…Target genes were selected using a large expression database generated by in situ hybridisation in Xenopus laevis (Gawantka et al, 1998;and www. dkfz-heidelberg.de/abt0135/axeldb.…”

High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization

“…Large-scale in situ hybridizations have been performed on mouse intestine (Komiya et al, 1997), E9.5 embryos (Gitton et al, 2002;Neidhardt et al, 2000), and E9.5 and E10.5 embryos (Reymond et al, 2002), and mouse brain as well as on other species, such as Drosophila (Tomancak et al, 2002), Zebrafish (Kudoh et al, 2001), Xenopus (Gawantka et al, 1998), Medaka Fish (Quiring et al, 2004), Chick retina (Shintani et al, 2004), Ascidian (Mochizuki et al, 2003), Chicken embryos (Bell et al, 2004). A robotic workstation is available, but due to its larger filter pore size (35 μm) it cannot be used for small embryos, such as mammalian preimplantation embryos.…”