Defining Synphenotype Groups in Xenopus tropicalis by Use of Antisense Morpholino Oligonucleotides

Abstract

To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between −80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing “rescue” experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis. Genome sequencing projects have provided remarkable insights into the expression and regulation of many genes. For some species, such as the invertebrates Caenorhabditis elegans and Drosophila melanogaster, it has been possible to assign functions to these genes on a genome-wide scale. For the vertebrates, similar efforts are being made in mouse and zebrafish, but work in the former species is expensive and slow, and the zebrafish experienced a whole genome duplication event, so that some genes may have retained redundant functions. Here, this study uses antisense morpholino oligonucleotides (MOs) to show that the diploid amphibian Xenopus tropicalis provides a powerful alternative species. The authors have designed MOs to target sequences around the initiating AUG codons of 202 genes expressed during early development and confirmed that these function in a specific manner. About 65% of the MOs caused embryos to develop abnormally, and the authors have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes. Expression pattern analysis indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into vertebrate development and paves the way for a comprehensive MO-based analysis of gene function in X. tropicalis.