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.
Highlights
The results of genome sequencing projects and the extensive analyses of expressed sequence tags (ESTs) have provided remarkable insights into the expression and regulation of many genes
The former category allows the comparison of phenotypes caused by injection of morpholino oligonucleotide (MO) in X. tropicalis with those obtained by other approaches in other species, including mouse and zebrafish, as well as X. laevis
In an attempt to strike a balance between eliminating the gene product of interest and not causing non-specific effects, we used a dose of 30 ng MO in our experiments
Summary
The results of genome sequencing projects and the extensive analyses of expressed sequence tags (ESTs) have provided remarkable insights into the expression and regulation of many genes. ‘‘Reverse genetic’’ screens of these sorts have the advantages of speed (because one does not have to locate the mutated gene) and economy, and a similar high-throughput approach to the investigation of gene function in vertebrate embryos will be very important for a proper understanding of development and disease. Such an approach cannot be adopted in mammalian embryos, except when studying very early stages [7], because the embryos are inaccessible and the abilities of most reagents to inhibit gene function decline as the embryos grow. The zebrafish is not a tetrapod, and like other teleost fish it underwent a whole genome duplication event between 200 and 450 million years ago [9,10], so that some genes are likely to have retained at least partially redundant functions [11,12]
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