RNA_Seq analysis of localization along animal-vegetal axis of Danio rerio

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and two daughter cells with different fates. This phenomenon is required for the establishment of many biological processes and is particularly responsible for the great variety of cell types formed during developmentand requires strict timing and positional control. The key molecules determining the body plan are the mRNAs, of which many examples have already been discovered to be asymmetrically localized during oogenesis and embryogenesis in both the amphibian and fish models. However, our knowledge about evolutionary conservation or differences of localized mRNAs is still limited to a few candidates. Our goal has been to compare localization profiles along the animal-vegetal axis of mature eggs of four diverse models, Xenopus laevis, Danio rerio, Ambystoma mexicanum and Acipenser ruthenus using the spatial expression analysis method called TOMO-Seq. Surprisingly, we revealed RNAs that code for many known important genes such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models and sometimes even missing in their genomes. We determined the transcriptome distribution and found a poor correlation between the vegetally localized genes but a relatively good correlation between the animally localized genes. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model. We performed cryosectioning of oocytes along the animal-vegetal axis (first developmental axis, section A (first animal) to section E (last vegetal), followed by RNA-Seq to determine the localization profiles of RNAs. The method allowed for a complete view on RNA localization. Total RNA was extracted using Qiagen (Microkit) column based isolation. The concentration of RNA was measured using a spectrophotometer (Nanodrop 2000, Thermo Scientific), and the quality of RNA was assessed using a Fragment Analyzer (AATI, High Sensitivity RNA analysis kit). RNA profiles were generated using RNA-Sequencing in 4 replicates (each divided into five segments).