Analysis of embryonic development in the unsequenced axolotl: Waves of transcriptomic upheaval and stability

Abstract

The axolotl (Ambystoma mexicanum) has long been the subject of biological research, primarily owing to its outstanding regenerative capabilities. However, the gene expression programs governing its embryonic development are particularly underexplored, especially when compared to other amphibian model species. Therefore, we performed whole transcriptome polyA+ RNA sequencing experiments on 17 stages of embryonic development. As the axolotl genome is unsequenced and its gene annotation is incomplete, we built de novo transcriptome assemblies for each stage and garnered functional annotation by comparing expressed contigs with known genes in other organisms. In evaluating the number of differentially expressed genes over time, we identify three waves of substantial transcriptome upheaval each followed by a period of relative transcriptome stability. The first wave of upheaval is between the one and two cell stage. We show that the number of differentially expressed genes per unit time is higher between the one and two cell stage than it is across the mid-blastula transition (MBT), the period of zygotic genome activation. We use total RNA sequencing to demonstrate that the vast majority of genes with increasing polyA+ signal between the one and two cell stage result from polyadenylation rather than de novo transcription. The first stable phase begins after the two cell stage and continues until the mid-blastula transition, corresponding with the pre-MBT phase of transcriptional quiescence in amphibian development. Following this is a peak of differential gene expression corresponding with the activation of the zygotic genome and a phase of transcriptomic stability from stages 9 to 11. We observe a third wave of transcriptomic change between stages 11 and 14, followed by a final stable period. The last two stable phases have not been documented in amphibians previously and correspond to times of major morphogenic change in the axolotl embryo: gastrulation and neurulation. These results yield new insights into global gene expression during early stages of amphibian embryogenesis and will help to further develop the axolotl as a model species for developmental and regenerative biology.

The axolotl genome and the evolution of key tissue formation regulators

“…Functional analysis of axolotl development, physiology and regeneration is facilitated by the availability of tissue-and time-dependent gene expression profiles [28][29][30]36 . The axolotl genome provides a foundation for applying methods such as chromatin immunoprecipitation with sequencing (ChIP-seq) or assay for transposase-accessible chromatin using sequencing (ATAC-seq) to investigate the genomic basis of gene regulation during regeneration.…”

Advances in Decoding Axolotl Limb Regeneration

“…This approach should benefit from future functional experimentation to determine the extent to which activation of putative oncogenes drives aspects of limb regeneration. 2016 saw the publication of the most comprehensive transcriptome assembly for axolotl to date [42]. This work reported the gene expression over the full course of axolotl embryogenesis.…”

Integrative Analysis of Axolotl Gene Expression Data From Regenerative and Wound Healing Limb Tissues

“…Consequently, only very recently sequencing and assembly of axolotl genome was reported [32]. Therefore, proteomics, transcriptomics (RNA-Seq), and microarrays have been the primary, indispensable tools for investigating gene networks and pathways of axolotl's regenerative mechanisms [17,22,[33][34][35][36]. While microarrays can be used to design probes for any condition of interest, RNA-Seq facilitates the detection of a wider range of expression values at a singlenucleotide resolution as well as accurately detects highly expressed genes with no saturation effect that is typically observed in microarrays [22].…”