Nascent transcriptome reveals orchestration of zygotic genome activation in early embryogenesis

•Whole-embryo and regional EU-RNA-seq determines timing and spatial patterns of ZGA •Maternal-zygotic genes dominate transcriptional output during ZGA •Manipulation of translation and cell division reconciles regulatory mechanisms of ZGA •Timing of germ-layer-specific expression appears sequential in the blastula Early embryo development requires maternal-to-zygotic transition, during which transcriptionally silent nuclei begin widespread gene expression during zygotic genome activation (ZGA). 1 Vastenhouw N.L. Cao W.X. Lipshitz H.D. The maternal-to-zygotic transition revisited. Development. 2019; 146: dev161471https://doi.org/10.1242/dev.161471 Crossref PubMed Scopus (112) Google Scholar , 2 Jukam D. Shariati S.A.M. Skotheim J.M. Zygotic genome activation in vertebrates. Dev. Cell. 2017; 42: 316-332https://doi.org/10.1016/j.devcel.2017.07.026 Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar , 3 Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027 Crossref PubMed Scopus (346) Google Scholar ZGA is vital for early cell fating and germ-layer specification, 3 Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027 Crossref PubMed Scopus (346) Google Scholar ,4 Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation. Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-x Crossref PubMed Scopus (175) Google Scholar and ZGA timing is regulated by multiple mechanisms. 1 Vastenhouw N.L. Cao W.X. Lipshitz H.D. The maternal-to-zygotic transition revisited. Development. 2019; 146: dev161471https://doi.org/10.1242/dev.161471 Crossref PubMed Scopus (112) Google Scholar , 2 Jukam D. Shariati S.A.M. Skotheim J.M. Zygotic genome activation in vertebrates. Dev. Cell. 2017; 42: 316-332https://doi.org/10.1016/j.devcel.2017.07.026 Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar , 3 Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027 Crossref PubMed Scopus (346) Google Scholar , 4 Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation. Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-x Crossref PubMed Scopus (175) Google Scholar , 5 Pálfy M. Joseph S.R. Vastenhouw N.L. The timing of zygotic genome activation. Curr. Opin. Genet. Dev. 2017; 43: 53-60https://doi.org/10.1016/j.gde.2016.12.001 Crossref PubMed Scopus (39) Google Scholar However, controversies remain about whether these mechanisms are interrelated and vary among species 6 Lu X. Li J.M. Elemento O. Tavazoie S. Wieschaus E.F. Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition. Development. 2009; 136: 2101-2110https://doi.org/10.1242/dev.034421 Crossref PubMed Scopus (83) Google Scholar , 7 Syed S. Wilky H. Raimundo J. Lim B. Amodeo A.A. The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities. Proc. Natl. Acad. Sci. USA. 2021; 118 (e2010210118)https://doi.org/10.1073/pnas.2010210118 Crossref Scopus (14) Google Scholar , 8 Strong I.J.T. Lei X. Chen F. Yuan K. O'Farrell P.H. Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate mid-blastula transition events at a low nuclear-cytoplasmic ratio. PLoS Biol. 2020; 18 (e3000891)https://doi.org/10.1371/journal.pbio.3000891 Crossref PubMed Scopus (13) Google Scholar , 9 Edgar B.A. Kiehle C.P. Schubiger G. Cell cycle control by the nucleo-cytoplasmic ratio in early Drosophila development. Cell. 1986; 44: 365-372 Abstract Full Text PDF PubMed Scopus (248) Google Scholar , 10 Collart C. Allen G.E. Bradshaw C.R. Smith J.C. Zegerman P. Titration of four replication factors is essential for the Xenopus laevis midblastula transition. Science. 2013; 341: 893-896https://doi.org/10.1126/science.1241530 Crossref PubMed Scopus (152) Google Scholar and whether the timing of germ-layer-specific gene activation is temporally ordered. 11 Argelaguet R. Clark S.J. Mohammed H. Stapel L.C. Krueger C. Kapourani C.A. Imaz-Rosshandler I. Lohoff T. Xiang Y. Hanna C.W. et al. Multi-omics profiling of mouse gastrulation at single-cell resolution. Nature. 2019; 576: 487-491https://doi.org/10.1038/s41586-019-1825-8 Crossref PubMed Scopus (166) Google Scholar ,12 Hashimshony T. Feder M. Levin M. Hall B.K. Yanai I. Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer. Nature. 2015; 519: 219-222https://doi.org/10.1038/nature13996 Crossref PubMed Scopus (109) Google Scholar In some embryonic models, widespread ZGA onset is spatiotemporally graded, 13 Chen H. Good M.C. Imaging nascent transcription in wholemount vertebrate embryos to characterize zygotic genome activation. Methods Enzymol. 2020; 638: 139-165https://doi.org/10.1016/bs.mie.2020.03.002 Crossref PubMed Scopus (2) Google Scholar ,14 Chen H. Einstein L.C. Little S.C. Good M.C. Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo. Dev. Cell. 2019; 49: 852-866.e7https://doi.org/10.1016/j.devcel.2019.05.036 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar yet it is unclear whether the transcriptome follows this pattern. A major challenge in addressing these questions is to accurately measure the timing of each gene activation. Here, we metabolically label and identify the nascent transcriptome using 5-ethynyl uridine (5-EU) in Xenopus blastula embryos. We find that EU-RNA-seq outperforms total RNA-seq in detecting the ZGA transcriptome, which is dominated by transcription from maternal-zygotic genes, enabling improved ZGA timing determination. We uncover discrete spatiotemporal patterns for individual gene activation, a majority following a spatial pattern of ZGA that is correlated with a cell size gradient. 14 Chen H. Einstein L.C. Little S.C. Good M.C. Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo. Dev. Cell. 2019; 49: 852-866.e7https://doi.org/10.1016/j.devcel.2019.05.036 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar We further reveal that transcription necessitates a period of developmental progression and that ZGA can be precociously induced by cycloheximide, potentially through elongation of interphase. Finally, most ectodermal genes are activated earlier than endodermal genes, suggesting a temporal orchestration of germ-layer-specific genes, potentially linked to the spatially graded pattern of ZGA. Together, our study provides fundamental new insights into the composition and dynamics of the ZGA transcriptome, mechanisms regulating ZGA timing, and its role in the onset of early cell fating. Early embryo development requires maternal-to-zygotic transition, during which transcriptionally silent nuclei begin widespread gene expression during zygotic genome activation (ZGA). 1 Vastenhouw N.L. Cao W.X. Lipshitz H.D. The maternal-to-zygotic transition revisited. Development. 2019; 146: dev161471https://doi.org/10.1242/dev.161471 Crossref PubMed Scopus (112) Google Scholar , 2 Jukam D. Shariati S.A.M. Skotheim J.M. Zygotic genome activation in vertebrates. Dev. Cell. 2017; 42: 316-332https://doi.org/10.1016/j.devcel.2017.07.026 Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar , 3 Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027 Crossref PubMed Scopus (346) Google Scholar ZGA is vital for early cell fating and germ-layer specification, 3 Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027 Crossref PubMed Scopus (346) Google Scholar ,4 Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation. Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-x Crossref PubMed Scopus (175) Google Scholar and ZGA timing is regulated by multiple mechanisms. 1 Vastenhouw N.L. Cao W.X. Lipshitz H.D. The maternal-to-zygotic transition revisited. Development. 2019; 146: dev161471https://doi.org/10.1242/dev.161471 Crossref PubMed Scopus (112) Google Scholar , 2 Jukam D. Shariati S.A.M. Skotheim J.M. Zygotic genome activation in vertebrates. Dev. Cell. 2017; 42: 316-332https://doi.org/10.1016/j.devcel.2017.07.026 Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar , 3 Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027 Crossref PubMed Scopus (346) Google Scholar , 4 Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation. Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-x Crossref PubMed Scopus (175) Google Scholar , 5 Pálfy M. Joseph S.R. Vastenhouw N.L. The timing of zygotic genome activation. Curr. Opin. Genet. Dev. 2017; 43: 53-60https://doi.org/10.1016/j.gde.2016.12.001 Crossref PubMed Scopus (39) Google Scholar However, controversies remain about whether these mechanisms are interrelated and vary among species 6 Lu X. Li J.M. Elemento O. Tavazoie S. Wieschaus E.F. Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition. Development. 2009; 136: 2101-2110https://doi.org/10.1242/dev.034421 Crossref PubMed Scopus (83) Google Scholar , 7 Syed S. Wilky H. Raimundo J. Lim B. Amodeo A.A. The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities. Proc. Natl. Acad. Sci. USA. 2021; 118 (e2010210118)https://doi.org/10.1073/pnas.2010210118 Crossref Scopus (14) Google Scholar , 8 Strong I.J.T. Lei X. Chen F. Yuan K. O'Farrell P.H. Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate mid-blastula transition events at a low nuclear-cytoplasmic ratio. PLoS Biol. 2020; 18 (e3000891)https://doi.org/10.1371/journal.pbio.3000891 Crossref PubMed Scopus (13) Google Scholar , 9 Edgar B.A. Kiehle C.P. Schubiger G. Cell cycle control by the nucleo-cytoplasmic ratio in early Drosophila development. Cell. 1986; 44: 365-372 Abstract Full Text PDF PubMed Scopus (248) Google Scholar , 10 Collart C. Allen G.E. Bradshaw C.R. Smith J.C. Zegerman P. Titration of four replication factors is essential for the Xenopus laevis midblastula transition. Science. 2013; 341: 893-896https://doi.org/10.1126/science.1241530 Crossref PubMed Scopus (152) Google Scholar and whether the timing of germ-layer-specific gene activation is temporally ordered. 11 Argelaguet R. Clark S.J. Mohammed H. Stapel L.C. Krueger C. Kapourani C.A. Imaz-Rosshandler I. Lohoff T. Xiang Y. Hanna C.W. et al. Multi-omics profiling of mouse gastrulation at single-cell resolution. Nature. 2019; 576: 487-491https://doi.org/10.1038/s41586-019-1825-8 Crossref PubMed Scopus (166) Google Scholar ,12 Hashimshony T. Feder M. Levin M. Hall B.K. Yanai I. Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer. Nature. 2015; 519: 219-222https://doi.org/10.1038/nature13996 Crossref PubMed Scopus (109) Google Scholar In some embryonic models, widespread ZGA onset is spatiotemporally graded, 13 Chen H. Good M.C. Imaging nascent transcription in wholemount vertebrate embryos to characterize zygotic genome activation. Methods Enzymol. 2020; 638: 139-165https://doi.org/10.1016/bs.mie.2020.03.002 Crossref PubMed Scopus (2) Google Scholar ,14 Chen H. Einstein L.C. Little S.C. Good M.C. Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo. Dev. Cell. 2019; 49: 852-866.e7https://doi.org/10.1016/j.devcel.2019.05.036 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar yet it is unclear whether the transcriptome follows this pattern. A major challenge in addressing these questions is to accurately measure the timing of each gene activation. Here, we metabolically label and identify the nascent transcriptome using 5-ethynyl uridine (5-EU) in Xenopus blastula embryos. We find that EU-RNA-seq outperforms total RNA-seq in detecting the ZGA transcriptome, which is dominated by transcription from maternal-zygotic genes, enabling improved ZGA timing determination. We uncover discrete spatiotemporal patterns for individual gene activation, a majority following a spatial pattern of ZGA that is correlated with a cell size gradient. 14 Chen H. Einstein L.C. Little S.C. Good M.C. Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo. Dev. Cell. 2019; 49: 852-866.e7https://doi.org/10.1016/j.devcel.2019.05.036 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar We further reveal that transcription necessitates a period of developmental progression and that ZGA can be precociously induced by cycloheximide, potentially through elongation of interphase. Finally, most ectodermal genes are activated earlier than endodermal genes, suggesting a temporal orchestration of germ-layer-specific genes, potentially linked to the spatially graded pattern of ZGA. Together, our study provides fundamental new insights into the composition and dynamics of the ZGA transcriptome, mechanisms regulating ZGA timing, and its role in the onset of early cell fating.