Abstract
The mRNAs stored in eggs are crucial for embryogenesis. To address functions of maternal mRNAs, we recently reported the novel method MASK (maternal mRNA-specific knockdown), which we used to specifically knockdown maternal transcripts in the ascidian Ciona intestinalis Type A. In MASK, the cis element of a maternal gene is fused with eGFP or Kaede reporter gene, and the cassette is introduced into Ciona genome by transposon-mediated transgenesis. In eggs of the transgenic lines, the maternal expression of the gene whose cis element is used for driving the reporter gene is suppressed. The zygotic expression of the gene is not suppressed, suggesting that the MASK method can distinguish between maternal and zygotic functions of a gene. Here we investigated the cis and trans factors responsible for MASK results. In the ovaries in which knockdown of a maternal gene occurs, a number of antisense small RNAs are expressed that are complementary to the sequence of the knocked-down genes. We suspect that these antisense small RNAs are the factor responsible for MASK results. The antisense small RNAs have several features that are seen in PIWI-interacting RNAs (piRNAs), suggesting that MASK is likely to use a piRNA-mediated mechanism to knock down maternal mRNAs.
Highlights
The initiative cues for animal development are given by genetic substances that are stored in eggs
Requirement of reporter gene for the maternal-specific knockdown. We showed that both eGFP and Kaede can induce MASK22
We showed that the marker cassette could be changed to the one expressing DsRed, the tested vector used eGFP for knocking down the maternal gene (Fig. 1b)
Summary
The initiative cues for animal development are given by genetic substances that are stored in eggs. The zygotic expression of neither eGFP nor the targeted maternal gene (if it has zygotic expression in Ciona) is silenced by MASK This is a strong advantage for establishing mutant lines of genes that have crucial roles in both maternal and zygotic functions by a reverse genetic method, and for precisely distinguishing between maternal and zygotic functions of genes. The requirement of the vector element necessary to induce MASK must be characterized in order to know how much we can modify the vector for MASK without losing the activity to knockdown maternal gene It is unknown how the insertion of the MASK vectors into the Ciona genome leads to the suppression of the maternal expression of endogenous genes. The uncertain activation of MASK is a serious disadvantage of this method, because screening for an appropriate transgenic line that shows efficient knockdown of the target gene must be conducted
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