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

Abstract

The diploid frog X. tropicalis has recently been adopted as a model genetic system, but loss-of-function screens in Xenopus have not yet been performed. We have undertaken a pilot functional knockdown screen in X. tropicalis for genes involved in nervous system development by injecting antisense morpholino (MO) oligos directed against X. tropicalis mRNAs. Twenty-six genes with primary expression in the nervous system were selected as targets based on an expression screen previously conducted in X. laevis. Reproducible phenotypes were observed for six and for four of these, a second MO gave a similar result. One of these genes encodes a novel protein with previously unknown function. Knocking down this gene, designated pinhead, results in severe microcephaly, whereas, overexpression results in macrocephaly. Together with the early embryonic expression in the anterior neural plate, these data indicate that pinhead is a novel gene involved in controlling head development. Developmental Dynamics 229:289 -299, 2004.

Simple vertebrate models for chemical genetics and drug discovery screens: Lessons from zebrafish and Xenopus

“…Alternatively, the screening of annotated chemical libraries can identify novel targets as demonstrated for matrix metalloproteinases, which were recently shown to play an essential role in pigment cell migration (Tomlinson et al, 2009a). Finally, large-scale morpholino knockdown screens (Kenwrick et al, 2004;Rana et al, 2006), genetic screens by random mutagenesis in Xenopus tropicalis (Goda et al, 2006), and novel targeted mutagenesis approaches (see Future Directions section) will make in vivo functional analysis of disease pathways and therapeutic target identification in this vertebrate model organism an even more attractive alternative to expensive and time-consuming mouse models.…”

A newt's eye view of lens regeneration

“…morpholinos are now possible (Henry and Elkins, 2001;Hirsh et al, 2002;Kenwrick et al, 2004). A thorough screening in lens regeneration might identify mutants and subsequently the gene(s) involved.…”

Xenomics

“…In addition, aMOs complementary to splice junction inhibit splicing of premRNA in vivo, providing another means of inhibiting gene function in the embryo (Kenwrick et al 2004;Sivak et al 2005). Targeting aMOs to splice junctions has the additional benefit that its effect can be monitored by RT-PCR (Kenwrick et al 2004;Sivak et al 2005). The disadvantage in targeting aMOs to splice junctions is that it requires knowledge of the genomic structure of the gene in question.…”

“…It was also shown that aMOs can efficiently and specifically inhibit translation of mRNAs in X. tropicalis into the tadpole stages (Nutt et al 2001). In addition, aMOs complementary to splice junction inhibit splicing of premRNA in vivo, providing another means of inhibiting gene function in the embryo (Kenwrick et al 2004;Sivak et al 2005). Targeting aMOs to splice junctions has the additional benefit that its effect can be monitored by RT-PCR (Kenwrick et al 2004;Sivak et al 2005).…”

“…However, given the extent of genome information now available in X. tropicalis (http://genome.jgi-psf.org/; http://www.ensembl.org/ Xenopus_tropicalis/index.html), it is now possible to identify splice junctions to most genes. Given that only sequence information around the start of translation and/or around splice junctions is needed to design aMOs, it has become possible to consider performing mid-to large-scale loss-of-function screens in X. tropicalis (Kenwrick et al 2004). Such an approach is likely to be very fruitful in the future for addressing the function of genes during early development on a large scale.…”