Fig. 8

Validation of maternal ACD transcript levels in oocytes and zygotes. Expression levels in FPKM at 2 hpf of selected ACD components in P. dumerilii zygotes based on RNA-seq are shown on the left. WMISH of selected ACD transcripts in mature oocytes/unfertilized eggs (0 hpf; upper row) and zygotes (2 hpf; lower row) are shown on the right. Importantly, WMISH procedures including timing of substrate development and washes were performed simultaneously for 0 and 2 hpf specimen for each individual probe to ensure comparable staining intensities between both stages. In oocytes, transcripts are concentrated in the clear cytoplasm surrounding the oocyte pronucleus. In zygotes transcripts are localized in the clear cytoplasm at the animal pole after ooplasmic segregation (compare to Fig. 1). Every tested gene exhibits similar or higher expression levels in oocytes than zygotes validating the RNA-seq data at 2hpf, and confirming the high maternal contribution for each ACD transcript. Background control WMISH using sense probes for the two highest expressed genes pcd11x and l(2)gl did not show any staining in early and late stages (Additional file 4: Figure S2)