Abstract
The maternal contribution to the oocyte cytoplasm plays an important role during embryogenesis because it is involved in early cell fate specification and embryonic axis establishment. However, screening projects targeting maternal factors have only been conducted in a limited number of animal models, such as nematodes, fruit flies, and zebrafish, while few maternal genes have been analysed because of difficulties encountered in inhibiting gene products already expressed in the ovaries. Therefore, simple and efficient methods for large-scale maternal screening are necessary. The appendicularian Oikopleura dioica is a planktonic tunicate member of the chordates. Gonadal microinjection and a novel gene knockdown method, DNA interference (DNAi), have been developed for use in this animal with the aim of inhibiting gene functions during oogenesis within the gonad. In this study, we adapted these methods for large-scale maternal factor screening, and observed malformation phenotypes related to some maternal factors. Approximately 2000 (56.9%) ovary-enriched gene products were screened, of which the knockdown of seven encoding genes resulted in various abnormalities during embryonic development. Most of these were related to microtubules and cell adhesion-related proteins. We conclude that DNAi is a potentially powerful screening tool for the identification of novel maternal factors in chordates.
Highlights
Oikopleura dioica is a planktonic tunicate belonging to the chordate phylum
The efficiency of DNA interference (DNAi) in the knockdown of maternal genes was indirectly shown for exogenously injected mRNA encoding a fluorescent protein[18]; there is no direct evidence for endogenous maternal genes
We carried out cDNA screening of maternal factors in the chordate O. dioica
Summary
Oikopleura dioica is a planktonic tunicate belonging to the chordate phylum. It is used as a model organism in developmental biology because of a number of advantageous traits: (i) a small number of constituent cells and invariant cell lineages, (ii) a rapid development and short life cycle of 5 days, (iii) a transparent embryo and adult body, (iv) a compact genome of 70 Mb, and (v) publicly available genome browser and transcriptomic microarray data[15,16]. DNA interference (DNAi) is another advantageous method that has been devised for use in O. dioica It involves the injection of PCR products to induce gene knockdown in a sequence-dependent manner[19]. This is a simple and inexpensive method for gene product screening because the preparation of PCR products is more straightforward than that of double strand RNA for RNA interference[17] These techniques make O. dioica a suitable animal for analysing the function of maternal factors because their function can be inhibited during oogenesis[19]. 2000 ovary-enriched genes were screened, and seven maternal factors were identified to induce various malformations of early embryos when inhibited These factors encode proteins involved in cell cleavage, nuclear transportation, cell–cell adhesion, and DNA replication. O. dioica is a useful experimental model for the analysis of maternal factor functions in chordates
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