Taylor N. Ayers, Matthew L. Nicotra, Miler T. Lee
Embryogenesis requires coordinated gene regulatory activities early on that establish the trajectory of subsequent development, during a period called the maternal-to-zygotic transition (MZT). The MZT comprises transcriptional activation of the embryonic genome and post-transcriptional regulation of egg-inherited maternal mRNA. Investigation into the MZT in animals has focused almost exclusively on bilaterians, which include all classical models such as flies, worms, sea urchin, and vertebrates, thus limiting our capacity to understand the gene regulatory paradigms uniting the MZT across all animals. Here, we elucidate the MZT of a non-bilaterian, the cnidarian Hydractinia symbiolongicarpus. Using parallel poly(A)-selected and non poly(A)-dependent RNA-seq approaches, we find that the Hydractinia MZT is composed of regulatory activities similar to many bilaterians, including cytoplasmic readenylation of maternally contributed mRNA, delayed genome activation, and separate phases of maternal mRNA deadenylation and degradation that likely depend on both maternally and zygotically encoded clearance factors, including microRNAs.
Across sexually reproducing organisms, the earliest stages of embryonic development are guided by cellular components inherited from the egg, including a large maternal RNA contribution. Eventually, new RNA are transcribed from the embryonic genome that will supplant the maternal RNA and assume developmental control as the embryo proceeds through stem cell induction and gastrulation. This maternal-to-zygotic transition (MZT) comprises two activities: the activation of the embryonic genome (zygotic genome activation, ZGA) and the removal of the maternal RNA contribution (maternal clearance), which combine to reprogram the embryonic transcriptome away from an egg identity to an embryonic stem cell identity . The regulatory logic and molecular bases for these processes remain to be fully deciphered.
Materials and method
H. symbiolongicarpus colonies were maintained and spawned as described in . Gametes were collected from breeder colonies 291–10 and 295–8 and used for fertilization in artificial sea water (Instant Ocean Reef Crystals ~28–31 ppt) at 20° C in a petri dish, reserving a portion of unfertilized eggs for immediate collection. After 15 minutes, fertilized eggs were washed, then the first time point was collected 15 minutes later while still at 1-cell stage (30 min post fertilization, m.p.f.). Embryos were transferred to a 23° C incubator for subsequent collections. For each collection, approximately 100 embryos were transferred into a 1.5 ml eppendorf tube, excess water removed, flash frozen in liquid nitrogen, and stored at -80° C until RNA extraction.
The Hydractinia maternal mRNA contribution undergoes extensive poly(A) tail length changes, prior to genome activation
To determine the timing of genome activation in Hydractinia, we needed to ensure that we could distinguish changes in absolute mRNA levels from changes in poly(A) tail length in the early embryo. To this end, we designed custom H. symbiolongicarpus antisense oligomers for rRNA-depletion RNA-seq, with the aid of our Oligo-ASST tool , targeting empirically determined nuclear and mitochondrial rRNA sequences (S1A–S1D Fig and S1, S2 Tables). We then performed RNA-seq on unfertilized eggs, fertilized eggs (0.5 h.p.f.), and embryos every hour from 1 to 7 h.p.f. at 23°C, spanning the onset of gastrulation  (Fig 1A). Both rRNA-depleted and poly(A)+ libraries were built in parallel for each sample. As a reference for later development, we additionally constructed poly(A)+ libraries during larval stages, 24, 48, and 72 h.p.f.
In sum, our transcriptome profile of early H. symbiolongicarpus embryogenesis has revealed strong thematic similarities in the maternal-to-zygotic transition between a cnidarian and bilaterians (Fig 7). Embryonic genome activation is preceded by a period of transcriptional quiescence, during which maternally contributed mRNA are either up-regulated via readenylation, or down-regulated via deadenylation as part of the first phase of maternal clearance. After genome activation, subsequent phases of maternal clearance are triggered, potentially involving de novo transcribed miRNAs. Thus, cytoplasmic polyadenylation, delayed genome activation, and maternal mRNA clearance are shared features of the MZT in both non-bilaterian and bilaterian animals. However, evidence for unusual embryonic histone and chromatin regulation likely involving H4K20 methylation so far distinguishes the Hydractinia MZT and warrants further investigation.
We thank U. Frank, Febrimarsa, and C. Schnitzler for discussions and advice, P. Rangan and T. Levin for feedback on the manuscript, and S. Sanders for assistance with animal collection. This project used the University of Pittsburgh Health Sciences Core at UPMC Children’s Hospital Pittsburgh for sequencing, and was supported by the University of Pittsburgh Center for Research Computing for computational resources.
Citation: Ayers TN, Nicotra ML, Lee MT (2023) Parallels and contrasts between the cnidarian and bilaterian maternal-to-zygotic transition are revealed in Hydractinia embryos. PLoS Genet 19(7): e1010845. https://doi.org/10.1371/journal.pgen.1010845
Editor: Mary C. Mullins, University of Pennsylvania Perelman School of Medicine, UNITED STATES
Received: May 11, 2023; Accepted: June 26, 2023; Published: July 13, 2023
Copyright: © 2023 Ayers et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Sequencing data are available in the Gene Expression Omnibus (GEO) under accession number GSE232065. Additional data files including the de novo assembled transcriptome are available at OSF, https://osf.io/jpq3v/.
Funding: M.T.L was supported by NIH grant R35GM137973. M.L.N. was supported by NSF grants 1557339 and 1923259. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.