Isolation of novel cDNAs by subtractions between the anterior mesendoderm of single mouse gastrula stage embryos

The anterior mesendoderm of mid- to late primitive streak stage mouse embryos has the ability to induce anterior neuroectodermal fate in naive epiblast [S.-L. Ang and J. Rossant (1993) Development 118, 139-149]. A number of genes have been found to be expressed in this tissue, notably the transcription factor Lim1. Lim1-null mice have anterior mesendoderm defects that result in a lack of head formation. Thus, the anterior mesendoderm of gastrula stage mouse embryos should express Lim1-regulated genes that are essential for head development. To identify Lim1-regulated genes, a differential screen with subtraction was developed, using cDNA pools that were amplified from the anterior mesendoderm of single wild-type and Lim1- null gastrula stage embryos. This novel screen strategy has yielded 22 cDNAs that show differential expression between anterior mesendoderm cells of wild- type and Lim1-null embryos. The expression of one novel cDNA SII6 initially colocalizes with Lim1 in the anterior mesendoderm of gastrula stage embryos. Moreover, SII6 expression is undetectable in the anterior mesendoderm of Lim1-null embryos. This screen identifies a set of putative Lim1 target genes that may have important roles in vertebrate head formation. Furthermore, this differential screen strategy should provide a broadly applicable approach to identify differences in gene expression between embryonic tissues of limiting quantity.