ABSTRACT In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.

ABSTRACT Elvan Böke studied molecular biology and genetics at the Middle East Technical University in Ankara, Turkey. She then moved to the Cancer Research UK Manchester Institute (CRUK-MI) for a PhD with Iain Hagan, where she discovered that a PP1–PP2A phosphatase relay promotes mitotic exit in fission yeast. For her postdoc, she moved to the lab of Tim Mitchison at Harvard Medical School, Boston, USA, where she unraveled how the enigmatic Balbiani body is formed by an amyloid-like assembly mechanism in oocytes. Elvan established her group at the Centre for Genomic Regulation (CRG) Barcelona, Spain, in 2017, where her lab investigates oocyte biology and cellular dormancy. She was awarded an ERC Starting Grant in 2017 and an ERC Consolidator Grant in 2022. In 2021, Elvan became a member of the EMBO Young Investigator Network.

ABSTRACT Lena Ho studied biochemistry at the University of Wisconsin–Madison, USA, funded by a scholarship from the Singapore Economic Development Board. Lena then joined the lab of Gerald Crabtree at Stanford University for her PhD, where she investigated the function and mechanism of chromatin remodelling proteins in the epigenetic regulation of embryonic, haematopoietic and cancer stem cells. She then moved back to Singapore for a postdoc in Bruno Reversade's lab at the Institute of Medical Biology, A*STAR, where she found that some non-coding RNAs can in fact encode short peptides, exemplified by her discovery of the peptide hormone ELABELA. In 2017, Lena became Assistant Professor at Duke–NUS Medical School and the Institute of Medical Biology, A*STAR, where her Endogenous Peptides lab currently focuses on secreted and mitochondria-localised peptides to uncover new paradigms in cell biology and improve cardiometabolic and immune health.

Lydia Finley studied biology at Yale University, USA, and then carried out her PhD work in the group of Marcia Haigis at Harvard Medical School on the post-translational regulation of mitochondrial function. During her postdoc in the lab of Craig Thompson at Memorial Sloan Kettering Cancer Center (MSKCC), she discovered that intracellular levels of the metabolite a-ketoglutarate, through regulating chromatin and gene expression, contribute to embryonic stem cell renewal. Lydia established her group at MSKCC in 2017, where they use genetic and metabolomic approaches to investigate the mechanisms that link metabolic pathways to cell fate decisions. Lydia is a Searle Scholar and the recipient of the Dale F. Frey Award for Breakthrough Scientists from the Damon Runyon Cancer Research Foundation.

Christian Munch studied biochemistry at the University of Tubingen and the Max Planck Institutes in Martinsried and Tubingen, Germany. For his PhD, he joined the group of Anne Bertolotti at the MRC Laboratory of Molecular Biology, Cambridge, UK, to work on protein aggregation and prion-like processes in neurodegenerative diseases. Then, he moved to Harvard Medical School, Boston, USA, to carry out postdoctoral work inWade Harper's lab, where he studied cellular protein quality control and the mitochondrial unfolded protein response. Christian established his independent research group in 2016 at the Institute of Biochemistry II, Goethe University Frankfurt, funded by an European Research Council (ERC) starting grant and Emmy Noether grant. His lab studies cellular stress responses to mitochondrial protein misfolding, infection and disease, as well as developing proteomics methods.

Amid the Coronavirus Disease 2019 (COVID-19) pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press, and policymakers for the first time. This phenomenon has sharpened long-standing questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through April 30, 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper.

s represent the first port of call for most readers, usually being freely available, brief, relatively 264 jargon-free, and machine-readable. Importantly, abstracts contain the key findings and conclusions 265 from an article. To analyse differences in abstracts between preprint and paper, we employed multiple 266 approaches. We first objectively compared textual changes between abstract pairs using a 267 computational approach before manually annotating abstracts (Fig. 3). Both approaches 268 demonstrated that COVID-19 articles underwent greater textual changes in their abstracts compared 269 to non-COVID-19 articles. However, in determining the type of changes, we discovered that 6% of non270 COVID-related abstracts and 15% of COVID-related abstracts had discrete, “major” changes in their 271 conclusions. Indeed, 42% of non-COVID-19 abstracts underwent no meaningful change between 272 preprint and published versions, though only 34% of COVID-19 abstracts were similarly unchanged. 273 The majority of changes were “minor” textual alterations that lead to a minor change or strengthening 274 or softening of conclusions. Of note, about 1/3 of changes were additions of new data (Fig 3F). While 275 previous works have focused their attention on the automatic processing of many other aspects of 276 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 20, 2021. ; https://doi.org/10.1101/2021.02.20.432090 doi: bioRxiv preprint

Stefanie Redemann studied Biology at Darmstadt Technical University, followed by a Master's at EMBL in Heidelberg, Germany. She then pursued a PhD in the labs of Tony Hyman and Jonathon Howard at the Max Planck Institute of Cell Biology and Genetics in Dresden, where she investigated the role of the actomyosin cortex in force generation and spindle positioning. After obtaining her doctorate degree in 2009, she joined the lab of Thomas Muller-Reichert at the Medical Theoretical Center in Dresden to work on reconstructing the mitotic spindle using electron tomography. Stefanie started her independent research group in 2018 at the University of Virginia School of Medicine, where she is using interdisciplinary approaches to study spindle assembly and chromosome segregation in both mitosis and meiosis.

ABSTRACT Tim Lämmermann studied molecular medicine at the Friedrich-Alexander-University, Erlangen-Nuremberg, Germany and the Lund University, Sweden. He then joined the lab of Michael Sixt at the Max Planck Institute of Biochemistry in Martinsried, where he earned his PhD in 2009 for studying the role of integrins and cytoskeletal forces in immune cell migration. Tim then moved to the National Institute of Allergy and Infectious Diseases in Bethesda, USA for his postdoc with Ron Germain. There, he worked on the mechanisms of neutrophil swarming during infection, and received the Robert-Koch Postdoctoral Award in 2014. Since 2015, Tim has been a Group Leader at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg, where his lab investigates the single-cell and population dynamics of immune cells. He was awarded an ERC Starting Grant in 2016.

The world continues to face a life-threatening viral pandemic. The virus underlying the COVID-19 disease, SARS-CoV-2, has caused over 98 million confirmed cases and 2.2 million deaths since January 2020. Although the most recent respiratory viral pandemic swept the globe only a decade ago, the way science operates and responds to current events has experienced a paradigm shift in the interim. The scientific community has responded rapidly to the COVID-19 pandemic, releasing over 125,000 COVID-19 related scientific articles within 10 months of the first confirmed case, of which more than 30,000 were hosted by preprint servers. We focused our analysis on bioRxiv and medRxiv, two growing preprint servers for biomedical research, investigating the attributes of COVID-19 preprints, their access and usage rates, as well as characteristics of their propagation on online platforms. Our data provides evidence for increased scientific and public engagement with preprints related to COVID-19 (COVID-19 preprints are accessed more, cited more, and shared more on various online platforms than non-COVID-19 preprints), as well as changes in the use of preprints by journalists and policymakers. We also find evidence for changes in preprinting and publishing behaviour: COVID-19 preprints are shorter and reviewed faster. Our results highlight the unprecedented role of preprints and preprint servers in the dissemination of COVID-19 science, and the impact of the pandemic on the scientific communication landscape. ### Competing Interest Statement JP is the executive director of ASAPbio, a non-profit organization promoting the productive use of preprints in the life sciences. GD is a bioRxiv Affiliate, part of a volunteer group of scientists that screen preprints deposited on the bioRxiv server. MP is the community manager for preLights, a non-profit preprint highlighting service. GD and JAC are contributors to preLights and ASAPbio Fellows. The authors declare no other competing interests.

Zdenek Lansky received his PhD in physics from the Charles University in Prague in 2006. He then moved to The Netherlands for his postdoc, where he studied the biophysics of molecular motors - first at the Vrije Universiteit in Amsterdam with Erwin Peterman, and then at Wageningen University with Marcel Janson. In 2011 he joined the lab of Stefan Diez at the B CUBE Center for Molecular Bioengineering in Dresden, where his research focused on crosslinker-mediated force generation in the cytoskeleton. Zdenek set up his lab at the Institute of Biotechnology, Czech Academy of Sciences, BIOCEV in 2015, which he has run together with Marcus Braun since 2017. Marcus Braun received his PhD in biochemistry from King's College London in 2009 for his work on microtubule-based molecular motors in the labs of Andrew McAinsh and Rob Cross. He then moved to Dresden for his postdoc to study cytoskeletal transport, with a particular focus on microtubule crosslinkers, in Stefan Diez's lab - first at the Max Planck Institute of Molecular Cell Biology and Genetics, then later at B CUBE. In 2017, he joined Zdenek Lansky at the Institute of Biotechnology, Czech Academy of Sciences, BIOCEV, as a principal investigator. The Lansky and Braun lab investigates ensemble dynamics of cytoskeletal proteins.

The world continues to face a life-threatening viral pandemic. The virus underlying the Coronavirus Disease 2019 (COVID-19), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has caused over 98 million confirmed cases and 2.2 million deaths since January 2020. Although the most recent respiratory viral pandemic swept the globe only a decade ago, the way science operates and responds to current events has experienced a cultural shift in the interim. The scientific community has responded rapidly to the COVID-19 pandemic, releasing over 125,000 COVID-19-related scientific articles within 10 months of the first confirmed case, of which more than 30,000 were hosted by preprint servers. We focused our analysis on bioRxiv and medRxiv, 2 growing preprint servers for biomedical research, investigating the attributes of COVID-19 preprints, their access and usage rates, as well as characteristics of their propagation on online platforms. Our data provide evidence for increased scientific and public engagement with preprints related to COVID-19 (COVID-19 preprints are accessed more, cited more, and shared more on various online platforms than non-COVID-19 preprints), as well as changes in the use of preprints by journalists and policymakers. We also find evidence for changes in preprinting and publishing behaviour: COVID-19 preprints are shorter and reviewed faster. Our results highlight the unprecedented role of preprints and preprint servers in the dissemination of COVID-19 science and the impact of the pandemic on the scientific communication landscape.

ABSTRACT Binyam Mogessie was born and raised in Ethiopia. He moved to Germany in 2004, where he studied biochemistry and cell biology at Jacobs University Bremen. He then moved to the UK for his PhD with Anne Straube, first at the Marie Curie Research Institute in Surrey and later at the Centre for Mechanochemical Cell Biology in Warwick, where he investigated the cellular mechanisms that organise the microtubule cytoskeleton during skeletal muscle differentiation. After receiving his PhD in cell biology from the University of London, he joined the laboratory of Melina Schuh in 2012 as a postdoc at the MRC-LMB in Cambridge (and later at the Max Planck Institute in Göttingen, Germany), where he discovered a function of the actin cytoskeleton in accurate chromosome segregation and the prevention of aneuploidy in mammalian eggs. Binyam established his independent research laboratory at the University of Bristol, School of Biochemistry in 2018, where he is a Wellcome Trust and Royal Society Sir Henry Dale fellow and HFSP Young Investigator. He also received a Seed Award from the Wellcome Trust and funding from the Rosetrees Trust and Royal Society. His lab is investigating actin- and microtubule-based cytoskeletal ensembles that promote healthy egg development and embryogenesis in mammals.

In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remain poorly understood. Here, we generated chromatin accessibility maps with ATAC-seq from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hypertranscribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. https://doi.org/10.7554/eLife.23326.001 eLife digest The DNA in a fertilized egg contains all the information required to form an animal’s body. In order for the animal to develop properly, particular genes encoded in the DNA are only active at specific times. The DNA is wrapped around proteins called histones, which allows the DNA to be tightly packed inside the cell. However, histones can block other proteins called transcription factors from binding to the DNA to activate the genes. Young embryos initially develop with all of their genes switched off, relying on the nutrients and other molecules provided by their mother. After some time, the embryo starts to switch on its own genes to take control of its own development, but it was not clear how this happens. Joseph et al. investigated how genes are activated in zebrafish embryos, which are often used as models to study how animals develop. The experiments show that competition between histones and transcription factors for binding to DNA controls when genes are switched on. In young fish embryos, there are so many histones present that transcription factors have no opportunity to bind to DNA. Over time, however, the numbers of histones decrease, allowing transcription factors to bind to DNA and switch on genes. Histones and transcription factors regulate the activity of genes throughout the life of the animal. Therefore, competition between these two types of protein may also control gene activity in other situations. A better understanding of how gene activity is controlled could allow researchers to more easily grow different types of cell in the laboratory or to reprogram specific cells in the body. As such, these new findings may aid the development of therapies to regenerate organs or tissues that have been damaged by injury or disease. https://doi.org/10.7554/eLife.23326.002 Introduction In many organisms, early embryonic development is directed exclusively by maternal products that are deposited into the female gamete during oogenesis. Following the clearance of a subset of these products (Yartseva and Giraldez, 2015), transcription is initiated and the zygotic genome acquires developmental control (Blythe and Wieschaus, 2015a; Harrison and Eisen, 2015; Lee et al., 2014; Tadros and Lipshitz, 2009). This handover is referred to as the maternal-to-zygotic transition and the onset of transcription is called zygotic genome activation (ZGA). The absolute time and number of cell cycles required before the first transcripts can be detected is species specific (Tadros and Lipshitz, 2009). Additionally, from one gene to another the timing of transcriptional activation varies (Aanes et al., 2011; Collart et al., 2014; Harvey et al., 2013; Heyn et al., 2014; Lott et al., 2011; Owens et al., 2016; Pauli et al., 2012; Sandler and Stathopoulos, 2016; Tan et al., 2013). In fact, for some genes the first zygotic transcripts can be detected several cell cycles before the stage that is traditionally defined as the time point of ZGA (De Renzis et al., 2007; Heyn et al., 2014; Skirkanich et al., 2011; Yang et al., 2002). In spite of the progress made, it remains unclear how the onset of transcription in embryos is temporally regulated. Several lines of evidence suggest that the absence of transcription during early embryonic development could be due to limited levels of transcription factors (Almouzni and Wolffe, 1995; Veenstra et al., 1999). In this scenario, transcriptional activation would occur once a threshold level of these factors is reached. For example, experiments that used the transcriptional activity of injected plasmids as a read-out revealed that an increase in the amount of the potent, heterologous, transcriptional activator GAL4-VP16 can overcome transcriptional repression of its target gene in the early embryo (Almouzni and Wolffe, 1995). However, it remained unclear whether limited levels of transcription factors contribute to the absence of endogenous transcription in early embryos. Additional support for the limited machinery model came from work showing that an increase in the concentration of the general transcription factor TBP can cause premature transcription from an injected – and incompletely chromatinized – DNA template in Xenopus embryos. This effect was maintained only when non-specific DNA was added to titrate chromatin assembly (Almouzni and Wolffe, 1995; Veenstra et al., 1999). These results suggested that low TBP levels may play a role in the absence of transcription during the early stages of Xenopus development, but that increasing TBP alone is not sufficient to cause sustained premature transcription. During the cleavage stages of Xenopus development, TBP levels increase due to translation, which suggests that TBP levels might contribute to the timely activation of transcription during ZGA (Veenstra et al., 1999). Transcription factors have recently been identified that are required for the activation of the first zygotically expressed genes in Drosophila (Zelda) and zebrafish (Pou5f3, Sox19b, Nanog) (Harrison et al., 2011; Lee et al., 2013; Leichsenring et al., 2013; Liang et al., 2008; Nien et al., 2011). RNA for these factors is maternally provided and their levels increase due to translation during the early cell cycles. This suggests the possibility that an increase in the concentration of these transcription factors might contribute to the shift from transcriptional repression to transcriptional activity. Although transcription factors levels clearly influence transcriptional activity during early embryogenesis, there is evidence to show that the transcriptional machinery is operational prior to ZGA (Dekens et al., 2003; Lu et al., 2009; Newport and Kirschner, 1982a, 1982b; Prioleau et al., 1994) (see below). Thus, the timing of ZGA cannot be solely explained by a requirement to reach a threshold level of transcriptional activators. The finding that a premature increase in the number of nuclei or the amount of DNA resulted in premature transcription of injected plasmids in Xenopus embryos suggested that the transcriptional machinery is fully functional prior to genome activation and led to the excess repressor model (Newport and Kirschner, 1982a). This model postulates that a transcriptional repressor is titrated by binding to the exponentially increasing amount of genomic DNA, until it is depleted first from the soluble fraction, and then from DNA, to allow for the onset of transcription. Related studies in zebrafish and Drosophila have provided further evidence for this model. Endogenous transcription is initiated earlier in zebrafish embryos that accumulate DNA due to a defect in chromosome segregation (Dekens et al., 2003), and transcription is delayed in haploid Drosophila embryos compared to diploid embryos, albeit not for all genes (Lu et al., 2009). The excess repressor model predicts that the repressor is present in large excess, at relatively stable levels while the genome is inactive, and can bind DNA with high affinity. Core histones fulfill these criteria (Adamson and Woodland, 1974; Woodland and Adamson, 1977). Moreover, when bound to DNA in the form of nucleosomes, histones can affect DNA accessibility for DNA-binding proteins. To date, two key studies have investigated the role of core histones in the temporal regulation of zygotic transcription in Xenopus embryos (Almouzni and Wolffe, 1995; Amodeo et al., 2015). Experiments that used the transcriptional activity of injected plasmids as a read-out revealed that premature transcription caused by an excess of non-specific DNA can be negated by the addition of histones (Almouzni and Wolffe, 1995). More recently, the level of histones H3/H4 was shown to regulate the level of transcription in Xenopus egg extract and H3 was suggested to play a similar role in the embryo (Amodeo et al., 2015). Taken together, these results support the idea that histones play a role in regulating the timing of zygotic transcription. If histones function as repressors according to the original excess repressor model, it would be predicted that a substantial reduction of the histone-density on DNA would cause the onset of transcription (Amodeo et al., 2015; Newport and Kirschner, 1982a). However, while such a scenario might be possible for typical sequence-specific repressors of transcription, it is unlikely for histones. Histones assemble into histone octamers on DNA to form nucleosomes, the basic building blocks of chromatin. Thus, random depletion of nucleosomes from DNA would severely compromise the integrity of chromatin structure. Taken together, there is support for the idea that histone levels play a role in regulating the timing of zygotic transcription, but it remains unclear how this would mechanistically work. Furthermore, the observation that both activator and histone levels play a role in shifting the balance between repression and activation at genome activation remains to be clarified. Here, we analyze the onset of zygotic transcription in zebrafish embryos. With a quantitative approach, we show that the concentration of non-DNA-bound histones determines the timing of zygotic transcription and that all four core histones are required for this effect. The reduction in nuclear histone concentration that coincides with genome activation does not result in a significant change in nucleosome density, but rather allows transcription factors to successfully compete for DNA binding. In agreement with this, the association of transcription factors with the genome is sensitive to histone levels, and changing the concentration of transcription factors also affects the time of transcription. Our results show that transcription is regulated by a dynamic competition for DNA binding between histones and transcription factors. Transcription begins when the concentration of non-DNA-bound histones in the nucleus has sufficiently dropped so that the transcriptional machinery can outcompete histones for binding to DNA. Results In zebrafish, zygotic transcription starts ~3 hr post-fertilization, around the tenth cell division (Aanes et al., 2011; Harvey et al., 2013; Heyn et al., 2014; Kane and Kimmel, 1993; Pauli et al., 2012) (Figure 1A). To analyze the onset of transcription in the embryo in detail, we identified six genes that are not maternally provided and that have previously been shown to be activated at the onset of genome activation (Aanes et al., 2011; Pauli et al., 2012) (Figure 1—figure supplement 1A). At the 1000-cell (1K) stage, transcripts can be detected, especially when choosing late stage embryos (Figure 1B). Thus, to clearly distinguish between transcription being off and on, we analyzed early 1K and (mid) high stage embryos. Using this approach, analysis by qPCR allowed us to detect consistent induction of these genes at high stage (Figure 1B and Figure 1—figure supplement 1B). We will refer to the stages before and after induction as before and following genome activation. To relate the onset of transcription to the number of cells present in the embryo, we next counted the number of cells in embryos ranging from 1K to dome stage. We imaged DAPI-stained nuclei on a two-photon microscope and counted them using the software Imaris (Figure 1C). Using nuclei as a proxy for cell number, we obtained counts that agreed with numbers of cells per embryo obtained by others for 1K (Keller et al., 2008). In contrast, our cell count for high stage, for example, was slightly higher (1900 vs 1800) (Keller et al., 2008), which is consistent with the later high stage which we analyzed (Figure 1D and Figure 1—source data 1). We conclude that the set of genes we selected is robustly induced at high stage, when embryos contain ~1900 cells, and thus represent a reliable system to analyze the onset of zygotic transcription during embryogenesis. Figure 1 with 1 supplement see all Download asset Open asset Assay to analyze the onset of transcription in zebrafish. (A) In zebrafish, transcription begins ~3 hr post-fertilization. Stage-specific drawings of representative embryos are adapted from Kimmel et al. (1995) with permission. (B) Expression of six genes was analyzed by qPCR at 512-cell, early 1K, mid 1K, high and oblong stage in wild-type embryos. Inset shows the same data, focusing on 1K and high stage. Data were taken from Figures 2B and 3D. Error bars represent SEM (n ≥ 4). (C) Cell counting of DAPI-stained nuclei imaged with a two-photon microscope. Multiple z-slices and four tiles were stitched to allow rendering in imaging software Imaris and the calculation of cell number. Image shown is a representative example of high stage. Scale bar, 100 μm. (D) Quantification of the number of cells at 1K, high, oblong, sphere and dome stage. Each data point represents a biological replicate consisting of three embryos. Error bars represent SEM (n = 3). (E) Western blots showing the protein level of histone H3, H4, H2A, and H2B in embryos at 8-cell, 64-cell, 256-cell and 1K stages. Tubulin was used to control for equal loading in each experiment. Blots shown are representative examples (n > 3). © 1995 John Wiley and Sons. All Rights Reserved. Figure 1 part A, lower panel sketches of embryos were reproduced from Kimmel et al. (1995) with permission. https://doi.org/10.7554/eLife.23326.003 Figure 1—source data 1 Cell numbers for wild-type zebrafish embryos. At every stage, cells were counted for three biological replicates, each consisting of three embryos. https://doi.org/10.7554/eLife.23326.004 Download elife-23326-fig1-data1-v2.xlsx Increasing the levels of all core histones delays onset transcription and gastrulation Experiments in Xenopus embryos led to the hypothesis that histone levels regulate the onset of zygotic transcription (Almouzni and Wolffe, 1995; Amodeo et al., 2015). To analyze whether in zebrafish, histones are potential candidates to be excess repressors, we analyzed the relative levels of the core histones—H3, H4, H2A and H2B—by Western blot. We found that they are present at relatively stable amounts from 8-cell to 1K stage (Figure 1E). Assuming that at 1K stage there are sufficient histones to wrap all genomes into chromatin, this suggests that histones are in excess relative to the amount of DNA during the earlier stages. Thus, histones could function as excess repressors of the zygotic genome in zebrafish. If histones function as excess repressors in zebrafish embryos, it would be predicted that their level would affect the onset of transcription. To test this, we analyzed the effect of increasing the amount of histones in the embryo on the timing of transcriptional activation. We injected a stoichiometric mixture of the four core histones (from here on referred to as histone cocktail, HC; see Materials and methods for more details) into embryos at the 1-cell stage and then analyzed the onset of transcription for the previously characterized set of genes (Figures 2A and 1B). An increase in the amount of histones delayed the onset of transcriptional activation: transcripts were detected at high stage in uninjected embryos, whereas in embryos injected with histone cocktail, transcription was only induced at oblong stage, a complete developmental stage later (Figure 2B and Figure 2—figure supplement 1A). Comparison of gene expression levels in uninjected and injected embryos at high stage (when transcripts can consistently be detected in uninjected embryos) revealed that the level of induction is reduced significantly upon injection of the histone cocktail but not upon injection of BSA as a control (Figure 2B and Figure 2—figure supplement 1A, bar graphs). Extending the analysis further, a large set of genes in Nanostring analysis confirmed that the effect we observed is general, and not limited to six genes (Figure 2C, Figure 2—figure supplement 2, Figure 2—source data 1). Staging by morphology was corroborated by cell counting, with absolute time between the analyzed stages being constant, confirming that changes in the timing of transcription were not due to effects on cell cycle length or developmental progression (Figure 2—figure supplement 1B). Moreover, the injected histones can be incorporated into chromatin, as indicated by labeling one of them with Cy5 and detecting this label in chromatin when imaging embryos after injection (Figure 2—figure supplement 1C), confirming that they are functional. Together, these data show that an increase in the excess amount of histones in the embryo delays the onset of transcription. Figure 2 with 2 supplements see all Download asset Open asset Increasing the levels of all core histones delays onset of transcription and gastrulation. (A) Schematic representation of experimental procedure. Histone cocktail (HC) containing ~5800 genomes worth of histones, or BSA was injected into the yolk of 1-cell embryos and qPCR and NanoString analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (B) Expression of mxtx2 and fam212aa was analyzed by qPCR at early 1K, high, and oblong stage in uninjected, BSA-injected and HC-injected embryos. Bar graphs show the same data, focusing on high stage. Error bars represent SEM (n ≥ 13). ***p<0.001 (two-tailed Student’s t test, compared to BSA control). (C) Expression of 53 zygotically expressed genes was analyzed by NanoString analysis at high stage in uninjected, BSA-injected and HC-injected embryos. Mean counts of three independent biological replicates are shown. Location of mxtx2 and fam212aa counts is indicated (See Figure 2—figure supplement 2 for more details). (D) Relative expression level of mxtx2 and fam212aa at high stage, for embryos injected with BSA, HC, and HC minus H3, H4, H2A, or H2B. Error bars represent SEM (n = 7). ***p<0.001 (ordinary one-way ANOVA). (E) Brightfield images of embryos that were not injected, injected with BSA, or injected with HC. Boxed images represent the onset of gastrulation. Scale bar shown for the uninjected 2-cell embryo applies to all treatments except for 24 hpf embryos which have a different scale bar. All scale bars represent 250 μm. hpf, hours post-fertilization. (F) Bar graph shows the quantification of the extra time it takes embryos to start gastrulation upon injecting BSA, HC, or HC minus one histone, compared to uninjected embryos. Error bars represent SEM (n = 27 for BSA, n = 25 for HC, n = 7 for HC minus one histone experiments). ***p<0.001 (ordinary one-way ANOVA with Tukey’s multiple comparison test). In B and D, mRNA levels are normalized to the expression of eif4g2α. https://doi.org/10.7554/eLife.23326.006 Figure 2—source data 1 NanoString probe set. https://doi.org/10.7554/eLife.23326.007 Download elife-23326-fig2-data1-v2.xlsx To test whether the effect we observe upon injecting the histone cocktail required an increase in the level of all four core histones, we next removed one histone at a time from the cocktail. The total protein content was kept constant by raising the level of the other three histones. Removing any histone from the histone cocktail impaired the ability of the histone cocktail to delay the onset of transcription (Figure 2D and Figure 2—figure supplement 1D). These results show that the injection of basic proteins into the embryo per se does not affect the onset of transcription. Moreover, these results argue that the effect of the histone cocktail relies on increasing the amount of all four histones and suggest that histones exert their repressive effect together. Since the onset of zygotic transcription is known to be required for gastrulation (Kane et al., 1996; Lee et al., 2013; Zamir et al., 1997), we analyzed the effect of injecting the histone cocktail on the onset of gastrulation. Embryos injected with the histone cocktail initiated gastrulation later than uninjected embryos (Figure 2E). Although there was a delay following injection of BSA, it was significantly shorter than that observed with the histone cocktail and appeared to be a non-specific effect of injection (Figure 2F). Following the onset of gastrulation, embryos appeared to develop normally (Figure 2E, 24 hpf). Removing any histone from the histone cocktail reduced the developmental delay we observed upon injecting the histone cocktail (Figure 2F). We note that the developmental delay in the minus-one histone experiments was not reduced to the level observed for BSA injections. We therefore expect that injecting histones has an additional effect on developmental progression that is independent of the delay in transcription. We conclude that the delay in transcription as a consequence of increased histone levels causes a delay in the onset of gastrulation. Decreasing the pool of available histones causes premature transcription If the level of histones regulates zygotic genome activation, it can also be predicted that a reduction in histone levels would result in the premature induction of transcription. A large fraction of the histones that is present at the onset of transcription is loaded in the egg already as protein (Figure 1E). Pentraxin three is a soluble pattern recognition molecule that has been shown to rapidly and irreversibly bind to the core histones H3 and H4 (Bottazzi et al., 2010; Daigo et al., 2014). We injected mRNA encoding PTX3 fused to RFP, to reduce the pool of available histones H3 and H4 in the zebrafish embryo (Figure 3A). As expected, total levels of H3 and H4 were not affected upon injection of this fusion construct (Figure 3B). Next, we examined if H4 co-precipitated with RFP-tagged PTX3 (Figure 3C). Indeed, this histone associates with PTX3 in vivo, suggesting that the injection of PTX3 results in a reduction of the soluble amount of histones H3 and H4 in zebrafish cells. A decrease in the soluble amount of histones caused premature transcription activation: transcripts were detected at early 1K stage, while in the uninjected embryos, transcripts were only detected at mid 1K (Figure 3D and Figure 3—figure supplement 1A). We included embryos at mid 1K in this experiment, in order to increase our resolution for detecting changes in transcription. Comparison of gene expression levels in uninjected and ptx3-injected embryos at early 1K stage (one time-point prior to when genes are first induced in uninjected embryos) revealed that the level of expression is increased upon injection of ptx3 mRNA (Figure 3D and Figure 3—figure supplement 1A, bar graphs). A control injection with rfp mRNA did not result in co-precipitation with H4 (Figure 3C), nor did it affect the onset of transcription (Figure 3D and Figure 3—figure supplement 1A). Staging by morphology was corroborated by cell counting (Figure 3—figure supplement 1B), with absolute time between the analyzed stages being constant. Taken together, our results provide evidence that the level of core histones in the embryo dictates the timing of transcriptional activation. Figure 3 with 1 supplement see all Download asset Open asset Decreasing the level of histones causes premature transcription. (A) Schematic representation of experimental procedure. ptx3-rfp or rfp (control) mRNA was injected into the cell of 1-cell embryos and qPCR analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (B) Western blot analysis of PTX3, histone H3 and H4 levels at 512-cell, 1K and high stage in uninjected embryos, rfp and ptx3-rfp mRNA-injected embryos. Tubulin was used to control for equal loading. Blots shown are representative examples (n = 3). (C) Western blot analysis for histone H4 after a pull-down using an RFP antibody at 1K stage. Uncoupled beads were used as a negative control. Blot shown is a representative example (n = 3). (D) Expression of mxtx2 and fam212aa was analyzed by qPCR at 512-cell, early 1K, and mid 1K stage in uninjected, rfp mRNA-injected and ptx3-rfp mRNA-injected embryos. Bar graphs focus on early 1K stage. Error bars represent SEM (n ≥ 4). *p<0.05; **p<0.01 (two-tailed Student’s t-test, compared to rfp mRNA control). mRNA levels are normalized to the expression of eif4g2α. https://doi.org/10.7554/eLife.23326.010 Onset of transcription coincides with a reduction in nuclear histone concentration If histones were to function as excess repressors according to the original excess repressor model, the concentration of non-DNA-bound histones would be predicted to decrease during the cleavage stages of development. To test this prediction, we determined the absolute (molar) content and, correspondingly, the number of molecules of core histones in embryos using a quantitative mass spectrometry approach we recently developed (Kumar et al., unpublished) (Figure 4A and Materials and methods for more details). We analyzed embryos ranging from 1-cell to shield stage, when gastrulation is well underway (Figure 4B and Figure 4—source data 1). We observed an increase in the levels of histone protein until 1K stage, after which levels remained reasonably stable until sphere stage. Then, a rapid increase was observed, which is most likely the result of translation of zygotically produced histone mRNAs. Knowing the absolute numbers of histones per embryo as well as the calculated number of histones required to wrap a genome (Figure 4B, Figre 4—source data 1), allowed us to derive the number of genomes worth of histones per embryo. From that, we derived the number of excess (non-DNA bound) histones per cell in genomes worth of histones, for embryos ranging from 1-cell to dome stage (Figure 4C and Materials and methods for more details). For example, at the 1-cell stage, there are 3098 times more histones per cell than are required to wrap the genome into chromatin. Due to the exponential increase in cell number during cleavage divisions, this number has dropped dramatically at 1K stage (Figure 4C). However, due to the large number of histones that is loaded in the oocyte, as well as the increase in histone level due to translation (Figure 4B), there are still nine genomes worth of non-DNA-bound histones per cell. Moreover, because what matters for protein-binding kinetics is the concentration, we next calculated the concentration of non-DNA-bound histones. Because the cleavage divisions are not accompanied by significant growth (the total animal cap volume increases by 29% from 128-cell to 1K, Figure 4—figure supplement 1A), the decreasing number of histones per cell is accompanied by a decreasing cellular volume, and the concentration of non-DNA bound histones in the cell does not change substantially (Figure 4D). Taken together, this shows that during transcription activation there is still a significant amount of non-DNA-bound histone and that the overall concentration of non-DNA-bound histones in the cell has not decreased by much. Figure 4 with 1 supplement see all Download asset Open asset Onset of transcription coincides with a reduction in nuclear histone concentration. (A) Our quantitative mass spectrometry approach. Zebrafish histones were quantified by comparing the abundances of native histone peptides with corresponding isotopically labeled peptides from the chimeric protein; chimeric protein was quantified by comparing the abundance of labeled (from chimera) and native (from standard) BSA peptides (see Materials and methods for more details). (B) Quantification of the number of histone H3, H4, H2A, and H2B per embryo at indicated stages by quantitative mass spectrometry. Error bars represent SEM (n = 3). (C) The excess number of histones per cell (in genomes worth) was calculated using H2B levels (Figure 4—source data 1) and cell numbers (Figure 1—source data 1), and by assuming an average of 1.5 genomes per cell (see Materials and methods for more details). For better visualization of the data at later developmental stages the values for 1-cell and 8-cell are not shown in the graph but are 3098 and 518, respectively. Error bars represent SEM (n = 3). GW, genomes worth of histones. (D) The total concentration of non-DNA-bound histones was calculated by dividing the excess genomes worth of histone H2B per embryo by the volume of the animal cap at the res

After fertilization, the embryonic genome is inactive until transcription is initiated during the maternal-to-zygotic transition. How the onset of transcription is regulated in a precisely timed manner, however, is a long standing question in biology. Several mechanisms have been shown to contribute to the temporal regulation of genome activation but none of them can fully explain the general absence of transcription as well the gene specific onset that follows. Here we review the work that has been done toward elucidating the mechanisms underlying the temporal regulation of transcription in embryos.

Cilia are cellular projections that perform sensory and motile functions. A key ciliary subdomain is the transition zone, which lies between basal body and axoneme. Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links. Here, we identify C. elegans CEP290 as a component of a third module required to form an inner scaffolding structure called the central cylinder. Co-inhibition of all three modules completely disrupted transition zone structure. Surprisingly, axoneme assembly was only mildly perturbed. However, dendrite extension by retrograde migration was strongly impaired, revealing an unexpected role for the transition zone in cell adhesion.