Abstract
O2 availability is one of the critical drivers of metazoan evolution and diversification. The earliest metazoans evolved in shallow marine shelves of the Neoproterozoic era, where the redox environment was likely variable and spatially heterogenous. This imposed physiological constraints on the emerging animals, selecting for O2-responsive and stress-adaptive traits. Embryogenesis is a novel and highly conserved stage of metazoan development, and its regulatory architecture may hold the key to understanding how adaptive traits arise in response to environmental change. Defining how early metazoan embryos respond to fluctuating O2 levels will therefore provide essential insights into the adaptive mechanisms that shaped the evolution of metazoans. Here, the embryos of the cnidarian Nematostella vectensis, a representative of early-diverging metazoans, were used to comprehensively investigate the developmental and genetic responses to hypoxia. N. vectensis embryogenesis is O2-dependent, with hypoxia inducing a reversible developmental arrest. Transcriptomic profiling reveals that the hypoxia response in N. vectensis embryos is conserved with bilaterians, encompassing core hypoxia-responsive genes and pathways. These findings suggest that the genetic toolkit underlying embryonic hypoxia responses was already established in the common cnidarian–bilaterian ancestor.
Similar content being viewed by others
Data availability
The sequencing data generated during this study have been deposited to the Sequence Read Archive (SRA), under accessions DRR794742-DRR794759. Code used for RNA-seq analyses is available upon request.
References
Lyons, T. W., Reinhard, C. T. & Planavsky, N. J. The rise of oxygen in Earth’s early ocean and atmosphere. Nature 506, 307–315 (2014).
Och, L. M. & Shields-Zhou, G. A. The Neoproterozoic Oxygenation Event: Environmental perturbations and biogeochemical cycling. Earth-Sci. Rev. 110, 26–57 (2012).
Dohrmann, M. & Wörheide, G. Dating early animal evolution using phylogenomic data. Sci. Rep. 7, 3599 (2017).
Lenton, T. M. & Daines, S. J. Biogeochemical transformations in the history of the ocean. Annu. Rev. Mar. Sci. 9, 31–58 (2017).
Erwin, D. H. et al. The Cambrian conundrum: Early divergence and later ecological success in the early history of animals. Science 334, 1091–1097 (2011).
Sperling, E. A., Knoll, A. H. & Girguis, P. R. The ecological physiology of Earth’s second oxygen revolution. Annu. Rev. Ecol. Evol. Syst. 46, 215–235 (2015).
Kaiho, K. et al. Oxygen increase and the pacing of early animal evolution. Glob. Planet. Change. 233, 104364 (2024).
Nursall, J. Oxygen as a prerequisite to the origin of the metazoa. Nature 183, 1170–1172 (1959).
Semenza, G. L. Life with oxygen. Science 318, 62–64 (2007).
Catling, D. C., Glein, C. R., Zahnle, K. J. & McKay, C. P. Why O2 is required by complex life on habitable planets and the concept of planetary“ oxygenation time’’. Astrobiology 5, 415–438 (2005).
Towe, K. M. Oxygen-collagen priority and the early metazoan fossil record. Proceedings of the National Academy of Sciences 65, 781–788 (1970).
Tostevin, R. & Mills, B. J. Reconciling proxy records and models of earth’s oxygenation during the neoproterozoic and palaeozoic. Interface focus 10, 20190137 (2020).
Sato, T. et al. Redox condition of the late neoproterozoic pelagic deep ocean: 57fe mössbauer analyses of pelagic mudstones in the ediacaran accretionary complex, Wales, UK. Tectonophysics 662, 472–480 (2015).
Stockey, R. G. et al. Sustained increases in atmospheric oxygen and marine productivity in the neoproterozoic and palaeozoic eras. Nature Geoscience 1–8 (2024).
Wei, G.-Y. et al. Global marine redox evolution from the late Neoproterozoic to the early Paleozoic constrained by the integration of Mo and U isotope records. Earth-Sci. Rev. 214, 103506 (2021).
Krause, A. J., Mills, B. J., Merdith, A. S., Lenton, T. M. & Poulton, S. W. Extreme variability in atmospheric oxygen levels in the late Precambrian. Sci. Adv. 8, eabm8191 (2022).
Brocks, J. J. et al. The rise of algae in cryogenian oceans and the emergence of animals. Nature 548, 578–581 (2017).
Hammarlund, E. U. et al. Benthic diel oxygen variability and stress as potential drivers for animal diversification in the Neoproterozoic-Palaeozoic. Nat. Commun. 16, 2223 (2025).
Mills, D. B. & Canfield, D. E. Oxygen and animal evolution: Did a rise of atmospheric oxygen “trigger’’ the origin of animals?. BioEssays 36, 1145–1155 (2014).
Hammarlund, E. U. Harnessing hypoxia as an evolutionary driver of complex multicellularity. Interface Focus 10, 20190101 (2020).
Bakleh, M. Z. & Al Haj Zen, A. The distinct role of hif-1α and hif-2α in hypoxia and angiogenesis. Cells 14, 673 (2025).
Dengler, F. Activation of AMPK under hypoxia: Many roads leading to Rome. Int. J. Mol. Sci. 21, 2428 (2020).
Bartoszewska, S. & Collawn, J. F. Unfolded protein response (UPR) integrated signaling networks determine cell fate during hypoxia. Cell. Mol. Biol. Lett. 25, 18 (2020).
Johannessen, M., Delghandi, M. P. & Moens, U. What turns CREB on?. Cell. Signal. 16, 1211–1227 (2004).
Hardie, D. G. & Ashford, M. L. AMPK: Regulating energy balance at the cellular and whole body levels. Physiology 29, 99–107 (2014).
Hollien, J. Evolution of the unfolded protein response. Biochimica et Biophysica Acta (BBA) 1833, 2458–2463 (2013).
Kalinka, A. T. et al. Gene expression divergence recapitulates the developmental hourglass model. Nature 468, 811–814 (2010).
von Abzhanov, A. Baer’s law for the ages: Lost and found principles of developmental evolution. Trends Genet. 29, 712–722 (2013).
Levin, M. et al. The mid-developmental transition and the evolution of animal body plans. Nature 531, 637–641 (2016).
Padilla, P. A. & Ladage, M. L. Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans. Cell Cycle 11, 1672–1679 (2012).
Foe, V. E. & Alberts, B. M. Reversible chromosome condensation induced in Drosophila embryos by anoxia: Visualization of interphase nuclear organization. J. Cell Biol. 100, 1623–1636 (1985).
DiGregorio, P. J., Ubersax, J. A. & O’Farrell, P. H. Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions. J. Biol. Chem. 276, 1930–1937 (2001).
Douglas, R. M., Xu, T. & Haddad, G. G. Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1555–R1563 (2001).
Padilla, P. A. & Roth, M. B. Oxygen deprivation causes suspended animation in the zebrafish embryo. Proceedings of the National Academy of Sciences 98, 7331–7335 (2001).
Gárriz, A. et al. Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas). Mol. Ecol. 31, 4319–4331 (2022).
Ryan, J. F. et al. The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science 342, 1242592 (2013).
Dunn, F. et al. A crown-group cnidarian from the Ediacaran of Charnwood Forest, UK. Nat. Ecol. Evol. 6, 1095–1104 (2022).
Thuesen, E. V. et al. Intragel oxygen promotes hypoxia tolerance of scyphomedusae. J. Exp. Biol. 208, 2475–2482 (2005).
Purcell, J. E. Pelagic cnidarians and ctenophores in low dissolved oxygen environments: a review. In Coastal hypoxia: consequences for living resources and ecosystems Vol. 58 77–100 (2001).
Wahl, M. The fluffy sea anemone Metridium senile in periodically oxygen depleted surroundings. Mar. Biol. 81, 81–86 (1984).
Putnam, N. H. et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86–94 (2007).
Fritzenwanker, J. H., Genikhovich, G., Kraus, Y. & Technau, U. Early development and axis specification in the sea anemone Nematostella vectensis. Dev. Biol. 310, 264–279 (2007).
Layden, M. J., Röttinger, E., Wolenski, F. S., Gilmore, T. D. & Martindale, M. Q. Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8, 924–934 (2013).
Wolenski, F. S., Layden, M. J., Martindale, M. Q., Gilmore, T. D. & Finnerty, J. R. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8, 900–915 (2013).
Karabulut, A., He, S., Chen, C.-Y., McKinney, S. A. & Gibson, M. C. Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis. Dev. Biol. 448, 7–15 (2019).
Mills, D. B. et al. The last common ancestor of animals lacked the hif pathway and respired in low-oxygen environments. eLife 7, e31176 (2018).
Tamulonis, C. et al. A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering. Dev. Biol. 351, 217–228 (2011).
Technau, U. Gastrulation and germ layer formation in the sea anemone Nematostella vectensis and other cnidarians. Mech. Dev. 163, 103628 (2020).
Zimmermann, B. et al. Topological structures and syntenic conservation in sea anemone genomes. Nat. Commun. 14, 8270 (2023).
Shao, Y., Wellman, T. L., Lounsbury, K. M. & Zhao, F.-Q. Differential regulation of GLUT1 and GLUT8 expression by hypoxia in mammary epithelial cells. Am. J. Physiol. Regul. Integr. Comp. Physiol. 307, R237–R247 (2014).
Kim, J.-W., Tchernyshyov, I., Semenza, G. L. & Dang, C. V. Hif-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia. Cell. Metab. 3, 177–185 (2006).
Yamaji, R. et al. Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: Involvement of Na⁺/Ca²⁺ exchanger. Biochimica et Biophysica Acta (BBA) 1593, 269–276 (2003).
Huang, L. et al. ALDOC and PGK1 coordinately induce glucose metabolism reprogramming and promote development of colorectal cancer. Mol. Med. 31, 239 (2025).
Chung, I.-C. et al. Unrevealed roles of extracellular enolase-1 (eno1) in promoting glycolysis and pro-cancer activities in multiple myeloma via hypoxia-inducible factor 1α. Oncol. Rep. 50, 205 (2023).
Krämer, A., Green, J., Pollard, J. Jr. & Tugendreich, S. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30, 523–530 (2014).
Lala, T. & Hall, R. A. Adhesion G protein-coupled receptors: Structure, signaling, physiology, and pathophysiology. Physiol. Rev. https://doi.org/10.1152/physrev.00027.2021 (2022).
Ling, C. et al. Ador-1 (adenosine receptor) contributes to protection against paraquat-induced oxidative stress in Caenorhabditis elegans. Oxid. Med. Cell. Longev. 2022, 1759009 (2022).
Sagi, D., de Lecea, L. & Appelbaum, L. Heterogeneity of hypocretin/orexin neurons. Front. Neurol. Neurosci. 45, 61 (2021).
Pozdniakova, S. & Ladilov, Y. Functional significance of the adcy10-dependent intracellular cAMP compartments. J. Cardiovasc. Dev. Dis. 5, 29 (2018).
Lory, P., Nicole, S. & Monteil, A. Neuronal Cav3 channelopathies: Recent progress and perspectives. Pflugers Arch. 472, 831–844 (2020).
Lin, S., Ke, M., Zhang, Y., Yan, Z. & Wu, J. Structure of a mammalian sperm cation channel complex. Nature 595, 746–750 (2021).
Sampieri, L., Di Giusto, P. & Alvarez, C. Creb3 transcription factors: ER-Golgi stress transducers as hubs for cellular homeostasis. Front. Cell Dev. Biol. 7, 123 (2019).
Liu, L. et al. Hypoxia-induced energy stress regulates mRNA translation and cell growth. Mol. Cell. 21, 521–531 (2006).
Alva, R., Wiebe, J. E. & Stuart, J. A. Revisiting reactive oxygen species production in hypoxia. Pflugers Arch. Eur. J. Physiol. 476, 1423–1444 (2024).
Acebal, M. C., Hansen, B. W., Jørgensen, T. S. & Dalgaard, L. T. Analysis of the transcriptional pathways associated with the induction of quiescent embryonic arrest in the calanoid copepod Acartia tonsa. Dev. Biol. 504, 38–48 (2023).
Houghton, F. D., Thompson, J. G., Kennedy, C. J. & Leese, H. J. Oxygen consumption and energy metabolism of the early mouse embryo. Mol. Reprod. Dev. 44, 476–485 (1996).
Miyazawa, H. & Aulehla, A. Revisiting the role of metabolism during development. Development https://doi.org/10.1242/dev.131110 (2018).
Cao, D. et al. Selective utilization of glucose metabolism guides mammalian gastrulation. Nature 634, 919–928 (2024).
Park, Y. Y., Ahn, J.-H., Cho, M.-G. & Lee, J.-H. ATP depletion during mitotic arrest induces mitotic slippage and APC/Cdh1-dependent cyclin B1 degradation. Exp. Mol. Med. 50, 1–14 (2018).
Chun, Y. & Kim, J. AMPK-mTOR signaling and cellular adaptations in hypoxia. Int. J. Mol. Sci. 22, 9765 (2021).
Walter, P. & Ron, D. The unfolded protein response: From stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011).
Li, J. et al. The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells. Cell Death Differ. 15, 1460–1471 (2008).
Díaz-Bulnes, P., Saiz, M. L., López-Larrea, C. & Rodríguez, R. M. Crosstalk between hypoxia and ER stress response: A key regulator of macrophage polarization. Front. Immunol. 10, 2951 (2020).
Liu, Y. et al. Regulation of G1 arrest and apoptosis in hypoxia by PERK and GCN2-mediated eIF2α phosphorylation. Neoplasia 12, 61-IN6 (2010).
Genikhovich, G. & Technau, U. Induction of spawning in the starlet sea anemone Nematostella vectensis, in vitro fertilization of gametes, and dejellying of zygotes. Cold Spring Harb. Protoc. 2009, pdb–prot5281 (2009).
Stefanik, D. J., Friedman, L. E. & Finnerty, J. R. Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8, 916–923 (2013).
Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Pertea, M. et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature biotechnology 33, 290–295 (2015).
Liao, Y., Smyth, G. K. & Shi, W. FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15550. (2014).
Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 10, 1523 (2019).
Acknowledgements
We thank J. Higuchi and A. Tanimoto for maintaining N. vectensis culture. We also appreciate the support from the OIST Sequencing (SQC), Scientific Imaging (IMG) and Scientific Computing and Data Analysis (SCDA) Sections.
Funding
This study was supported by JSPS KAKENHI Grant Number JP24KF0262 and Okinawa Institute of Science and Technology Graduate University.
Author information
Authors and Affiliations
Contributions
S.H. and H.Wang conducted the experiments and performed data analyses, Y.H. designed and constructed the hypoxia culture system, H.Watanabe conceived the research. S.H., H.Wang, and H.Watanabe wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Hadife, S., Wang, H., Hongo, Y. et al. Hypoxia-induced gene expression changes in N. vectensis embryos. Sci Rep (2026). https://doi.org/10.1038/s41598-026-44143-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-44143-x
