Identification of Early Zygotic Genes in the Yellow Fever Mosquito Aedes aegypti and Discovery of a Motif Involved in Early Zygotic Genome Activation

Abstract

During early embryogenesis the zygotic genome is transcriptionally silent and all mRNAs present are of maternal origin. The maternal-zygotic transition marks the time over which embryogenesis changes its dependence from maternal RNAs to zygotically transcribed RNAs. Here we present the first systematic investigation of early zygotic genes (EZGs) in a mosquito species and focus on genes involved in the onset of transcription during 2–4 hr. We used transcriptome sequencing to identify the “pure” (without maternal expression) EZGs by analyzing transcripts from four embryonic time ranges of 0–2, 2–4, 4–8, and 8–12 hr, which includes the time of cellular blastoderm formation and up to the start of gastrulation. Blast of 16,789 annotated transcripts vs. the transcriptome reads revealed evidence for 63 (P<0.001) and 143 (P<0.05) nonmaternally derived transcripts having a significant increase in expression at 2–4 hr. One third of the 63 EZG transcripts do not have predicted introns compared to 10% of all Ae. aegypti genes. We have confirmed by RT-PCR that zygotic transcription starts as early as 2–3 hours. A degenerate motif VBRGGTA was found to be overrepresented in the upstream sequences of the identified EZGs using a motif identification software called SCOPE. We find evidence for homology between this motif and the TAGteam motif found in Drosophila that has been implicated in EZG activation. A 38 bp sequence in the proximal upstream sequence of a kinesin light chain EZG (KLC2.1) contains two copies of the mosquito motif. This sequence was shown to support EZG transcription by luciferase reporter assays performed on injected early embryos, and confers early zygotic activity to a heterologous promoter from a divergent mosquito species. The results of these studies are consistent with the model of early zygotic genome activation via transcriptional activators, similar to what has been found recently in Drosophila.

Transcriptional Activation Is a Conserved Feature of the Early Embryonic Factor Zelda That Requires a Cluster of Four Zinc Fingers for DNA Binding and a Low-complexity Activation Domain

“…Supporting this conserved role of ZLD in driving genome activation in other insects, sequences similar to TAGteam elements (VBRGGTA, V ϭ A/C/G, B ϭ C/G/T, r ϭ A/G) are enriched in the 400-base pair regions upstream of the set of genes activated at the MZT in the mosquito Aedes aegypti (30). Comparison of position weight matrices for the A. aegypti sequence and D. melanogaster TAGteam elements suggests that these two sequence elements may be homologous, and, like the D. melanogaster TAGteam sites, a region containing this sequence is capable of driving early zygotic transcription (9,30).…”

“…Comparison of position weight matrices for the A. aegypti sequence and D. melanogaster TAGteam elements suggests that these two sequence elements may be homologous, and, like the D. melanogaster TAGteam sites, a region containing this sequence is capable of driving early zygotic transcription (9,30). Together, these data suggest that both the DNA-binding domain of ZLD and the sequence to which it binds are conserved in divergent species.…”

Zygotic Genome Activation During the Maternal-to-Zygotic Transition

“…Zelda binds promoter regions of early zygotic genes via a heptamer DNA motif called the TAGteam (De Renzis et al, 2007; ten Bosch et al, 2006) that is conserved in other insects (Biedler et al, 2012). Loss of Zelda causes mitotic defects (Staudt et al, 2006) and a failure to activate 120 early zygotic genes during cycles 8-13 (Liang et al, 2008).…”

MicroRNAs of two medically important mosquito species: Aedes aegypti and Anopheles stephensi

“…aegypti represent maternally deposited miRNAs as zygotic transcription does not commence until after ~2h post oviposition (Biedler and Tu, 2010; Biedler et al, 2012). We showed that 58 miRNAs have at least 5 raw counts, with 26 having more than 200 raw counts at the 0–1h time window (Table S1).…”

“…There is indication that gene expression during the maternal-to-zygotic transition may vary significantly between Ae. aegypti and Drosophila melanogaster (Biedler and Tu, 2010; Biedler et al, 2012). Thus we also performed a detailed analysis of miRNAs during maternal-to-zygotic transition in Ae.…”