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Embryonic development is controlled by a complex interaction between maternal and zygotic activities. Maternal messenger RNAs and proteins are deposited in the unfertilized egg during oogenesis; after fertilization, the activation of the zygotic genome is accompanied by the degradation of a fraction of maternally supplied transcript...
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... 3-fold cut-off identifies all mRNAs that are at least 67% supplied by zygotic transcription at cycle 14. Combining the data from all four manipulations, we estimate that such zygotically active genes represent about 18% of the genes detectable at cycle 14, i.e., 1,158 genes distributed on all four chromosomes (Table S1). The remaining mRNA species appear to be supplied predominantly by maternal tran- scription. ...
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... 646 cases, the maternal degradation was at least in part compensated by zygotic transcription (Table S7). A representative list of maternal and zygotic transcripts known to be degraded or induced during the MZT and detected by our analysis is shown in Table 1. ...
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... genes (including Snail, Zen, and Nullo, see Table S8 for a complete list) are expressed even prior to the gap and pair-rule genes, which in our measurements do not yet show significant increased levels at this time point. Expression of gap and pair- rule transcripts was detected at 2-3 h (Table S1), arguing that their transcripts accumulate with a slower kinetic. When searching for over-represented motifs in the 2-kb upstream regions of these genes, we found the same motif as for the pure zygotic genes, along with other overlapping or slightly distinct variants ( Figure 4B). ...
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... KB JPG). Table S1. List of Zygotic Genes: Primary List of mRNAs that were down-regulated at least 3-fold compared to similarly staged WT embryos in cycle 14, upon ablation of each chromosome, and that were located on the chromosome that was removed. ...
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... of Purely Zygotic Genes (Identified Using Chromosome Deletion þ Time Course) List of mRNAs that were not expressed at 0-1 h, were down-regulated at least 3-fold compared to similarly staged WT embryos in cycle 14, upon ablation of each chromosome, and that were located on the chromosome that was removed. Therefore, these are purely zygotic transcripts and represent a subgroup of Table S1. This last table includes both purely zygotic as well as maternalþzygotic. ...
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... The minor, early wave of ZGA in D. melanogaster begins at nuclear cycle 8 and concludes at 54 cycle 12 when the major, later wave begins and the remainder of the genome is activated (De 55 Renzis et al., 2007;Tadros & Lipshitz, 2009). Zygotic histone gene expression is first detectable 56 around nuclear cycle 11 (White et al., 2007). ...
To ensure that the embryo can package exponentially increasing amounts of DNA, replication-dependent histones are some of the earliest transcribed genes from the zygotic genome. However, how the histone genes are identified is not known. The pioneer factors Zelda and CLAMP collaborate at a subset of genes to regulate zygotic genome activation in Drosophila melanogaster and target early activated genes to induce transcription. CLAMP also regulates the embryonic histone genes and helps establish the histone locus body, a suite of factors that controls histone mRNA biosynthesis. The relationship between Zelda and CLAMP led us to hypothesize that Zelda helps identify histone genes for early embryonic expression. We found that Zelda targets the histone locus early during embryogenesis, prior to histone gene expression. However, depletion of zelda in the early embryo does not affect histone mRNA levels or histone locus body formation. While surprising, these results concur with other investigations into Zelda’s role in the early embryo, suggesting the earliest factors responsible for specifying the zygotic histone genes remain undiscovered.
... This window increases with each cycle. In concordance with the time constraint, the pre-MBT transcripts are short, lack introns, and show signs of being aborted Kwasnieski et al, 2019;De Renzis et al, 2007), with the potential explanation simply being that mitosis terminates transcription. This minor wave of zygotic transcription at nc 8-12 is followed by a major wave of zygotic transcription of approximately one-third of all genes at nc 14. ...
... 7 Maternal factors are degraded, and zygotic transcription takes over gradually through two waves of zygotic gene activation (ZGA). [8][9][10] The major wave of gene expression associated with ZGA coincides with the 14 th nuclear cycle (nc14), during which time cell membranes grow between the blastoderm nuclei and cellularization takes place. [11][12][13] Spatially localized gene transcription in the ventral domain (e.g., the expression of snail [sna] and twist [twi]) associated with the presumptive mesoderm at mid-nc14 is the first sign of lineage diversification during embryogenesis. ...
Single-cell technologies promise to uncover how transcriptional programs orchestrate complex processes during embryogenesis. Here, we apply a combination of single-cell technology and genetic analysis to investigate the dynamic transcriptional changes associated with Drosophila embryo morphogenesis at gastrulation. Our dataset encompassing the blastoderm-to-gastrula transition provides a comprehensive single-cell map of gene expression across cell lineages validated by genetic analysis. Subclustering and trajectory analyses revealed a surprising stepwise progression in patterning to transition zygotic gene expression and specify germ layers as well as uncovered an early role for ecdysone signaling in epithelial-to-mesenchymal transition in the mesoderm. We also show multipotent progenitors arise prior to gastrulation by analyzing the transcription trajectory of caudal mesoderm cells, including a derivative that ultimately incorporates into visceral muscles of the midgut and hindgut. This study provides a rich resource of gastrulation and elucidates spatially regulated temporal transitions of transcription states during the process.
... Five of such "cellularization genes" have been identified as follows: nullo, serendipity-α, bottleneck (bnk), slam, and dunk (Merrill et al., 1988;Wieschaus and Sweeton, 1988;Schweisguth et al., 1990;Rose and Wieschaus, 1992;Schejter and Wieschaus, 1993;Lecuit et al., 2002;He et al., 2016). The transition from the syncytial stage to cellularization is preceded by the massive induction of zygotic gene expression in the embryo (Renzis et al., 2007;Liang et al., 2008;Tadros and Lipshitz, 2009). The five cellular-ization genes are all expressed in a short time window around the onset of cellularization, and the corresponding proteins rapidly disappear during late cellularization. ...
Drosophila cellularization is a special form of cleavage that converts syncytial embryos into cellular blastoderms by partitioning the peripherally localized nuclei into individual cells. An early event in cellularization is the recruitment of non-muscle myosin II ("myosin") to the leading edge of cleavage furrows, where myosin forms an interconnected basal array before reorganizing into individual cytokinetic rings. The initial recruitment and organization of basal myosin are regulated by a cellularization-specific gene, dunk, but the underlying mechanism is unclear. Through a genome-wide yeast two-hybrid screen, we identified anillin (Scraps in Drosophila), a conserved scaffolding protein in cytokinesis, as the primary binding partner of Dunk. Dunk colocalizes with anillin and regulates its cortical localization during the formation of cleavage furrows, while the localization of Dunk is independent of anillin. Furthermore, Dunk genetically interacts with anillin to regulate the basal myosin array during cellularization. Similar to Dunk, anillin colocalizes with myosin since the very early stage of cellularization and is required for myosin retention at the basal array, prior to the well-documented function of anillin in regulating cytokinetic ring assembly. Based on these results, we propose that Dunk regulates myosin recruitment and spatial organization during early cellularization by interacting with and regulating anillin.
... Protein sequences of all single-copy gene families were retrieved from the gene family clustering analysis and used for the construction of a phylogenetic tree based on the 15 representative animal species. The protein sequences for each gene family were aligned using MUSCLE (Edgar, 2004) and gaps were trimmed using Gblocks (Talavera & Castresana, 2007). Then, a super alignment matrix was achieved through concatenating the alignments. ...
... Briefly, reference protein sequences of those target genes were retrieved from GenBank, and subsequently aligned to the protein sets of each species using MUSCLE (Edgar, 2004) with the default parameters. We only retained the best hits determined by the program in each species for further analysis. ...
... Variation in exon number was also detected in the three of rest four genes (Twist2, Rdh10, and SNAP25), suggesting that the variation in exon number of head circumference modulation-related genes highlighted the importance of those genes in regulating the development of large head in P. megacephalum, as inferred from the crucial role of variation in exon number in organ development or disease occurrence (Kim et al., 2020). The occurrence of intronless genes suggested high transcription efficiency due to no need of post-transcriptional splicing (De Renzis et al., 2007;Li et al., 2017). The utilization of intronless GAS1 in P. megacephalum may represent an adaptive change to enhance transcription efficiency to facilitate the large head. ...
The big-headed turtle (Platysternon megacephalum) is an endemic chelonian species in Asia. Unlike most other turtles in the world, P. megacephalum is characterized with eagle-beak jaw, large head, and long tail. Although these unique characteristics are well recognized, the underlying genetic basis remains largely elusive. Here, we performed comparative genomic analysis between P. megacephalum and other representative species, aiming to reveal the genetic basis of the unique morphological features. Our results revealed that the eagle-beak jaw is most likely enabled by combined effects of expansion of SFRP5, extraction of FGF11, and mutation of both ZFYVE16 and PAX6. Large head is supported by mutations of SETD2 and FGRF2 and copy number variations of six head ircumference modulation-related genes (TGFBR2, Twist2, Rdh10, Gas1, Chst11, and SNAP25). The long tail is probably involved in a genetic network comprising Gdf11, Lin 28, and HoxC12, two of which showed a consistent expression pattern with a model organism mice). These findings suggest that expansion, extraction, and utation of those genes may have profound effects on unique phenotypes of P. megacep
... To detect subtler signals of embryonic genome activation, we quantified intronic sequencing read coverage. In other animals, a large proportion of de novo transcription at genome activation occurs for genes that already have a maternal RNA contribution, which can be difficult to dissect apart in RNA-seq data [2,18,31,48,[57][58][59]. Previously, we demonstrated that RNA-seq reads mapping to intronic regions can detect de novo pre-mRNA production in maternal genes where increases in exonic read coverage are too modest to robustly quantify [18,59]. ...
... Genome activation in Hydractinia is widespread across thousands of genes that were also expressed in the maternal contribution (Fig 1B-1D), similar to other animals [2,18,31,48,59]; as well as repetitive and transposon-associated elements, reinforcing a growing trend of the roles of transposons in shaping early embryonic gene expression across taxa by contributing regulatory sequences that can be co-opted to drive early gene activation [69,[75][76][77]. However, it is the massive activation of histone gene transcription that underlies the major change to the blastula/gastrula transcriptome (Fig 2). ...
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. But we also observe massive upregulation of histone genes and an expanded repertoire of predicted H4K20 methyltransferases, aspects thus far particular to the Hydractinia MZT and potentially underlying a novel mode of early embryonic chromatin regulation. Thus, similar regulatory strategies with taxon-specific elaboration underlie the MZT in both bilaterian and non-bilaterian embryos, providing insight into how an essential developmental transition may have arisen in ancestral animals.
... To detect subtler signals of embryonic genome activation, we quantified intronic sequencing read coverage. In other animals, a large proportion of de novo transcription at genome activation occurs in genes that already have a maternal RNA contribution, which can be difficult to dissect apart in RNA-seq data (Chen and Good, 2022;De Renzis et al., 2007;Harvey et al., 2013;Heyn et al., 2014;Lee et al., 2014Lee et al., , 2013Phelps et al., 2022). Previously, we demonstrated that RNA-seq reads mapping to intronic regions can detect de novo pre-mRNA production in maternal genes where increases in exonic read coverage are too modest to robustly quantify (Lee et al., 2013;Phelps et al., 2022). ...
... Genome activation in Hydractinia is widespread across thousands of genes that were also expressed in the maternal contribution (Fig 1B-D), similar to other animals (De Renzis et al., 2007;Harvey et al., 2013;Lee et al., 2014Lee et al., , 2013Phelps et al., 2022); as well as repetitive and transposon-associated elements, reinforcing a growing trend of the roles of transposons in shaping early embryonic gene expression across taxa (Modzelewski et al., 2022). However, it is the massive activation of histone gene transcription that underlies the major change to the blastula/gastrula transcriptome (Fig 2). ...
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 analogous 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. But we also observe massive upregulation of histone genes and an expanded repertoire of predicted H4K20 methyltransferases, aspects thus far unique to the Hydractinia MZT and potentially underlying a novel mode of early embryonic chromatin regulation. Thus, similar regulatory strategies with taxon-specific elaboration underlie the MZT in both bilaterian and non-bilaterian embryos, providing insight into how an essential developmental transition may have arisen in ancestral animals.
... During the mid-blastula transition, as the supply of maternally deposited factors declines, zygotic transcription will commence in a process known as zygotic genome activation (ZGA) (1-3). The onset time varies depending on the organism (4)(5)(6)(7)(8). ZGA is generally thought to occur in two main phases, although a more continuous model of activation has also been proposed (9). ...
... ZGA is generally thought to occur in two main phases, although a more continuous model of activation has also been proposed (9). In the first minor wave, a few hundred genes are transcribed between nuclear cycles 8 and 13 (7)(8)(9)(10)(11). Those genes are usually short and intronless, and they rely on the pioneer transcription factor Zelda (5,7,9,12,13). ...
... In the first minor wave, a few hundred genes are transcribed between nuclear cycles 8 and 13 (7)(8)(9)(10)(11). Those genes are usually short and intronless, and they rely on the pioneer transcription factor Zelda (5,7,9,12,13). During the second major wave at nuclear cycle 14, about 6000 genes start to be transcribed in a narrow time window (7-9, 14, 15). ...
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. Here, we used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. Our screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. We demonstrate that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, our data identify Nejire and Gcn5 as key regulators of ZGA.
... Gene length also influences the differential timing of expression of the paralogous gap genes knirps (3 kb) and knirps-related (23 kb) (Rothe et al. 1992). Indeed, most of the genes active in precellular Drosophila embryos are quite small and often lack introns (De Renzis et al. 2007;Artieri and Fraser 2014), implying that gene length can be a critical structural element under selective pressure in developing embryos with fast cell cycles. ...
... We also examined the expression of genes that are activated during the minor and major waves of ZGA (59) and are involved in the first spatial patterning events of the embryo. The majority of these genes had no significant changes in their mRNA levels after the depletion of BEAF-32, CTCF, or CP190, with a few exceptions including the up-regulation of the homeotic gene scr in CTCF mutant embryos (table S3). ...
... After the depletion of each of the three factors, a few hundred zygotically expressed genes had significant changes in their expression (Fig. 5). However, it is interesting to note that this did not include many of the "classic" minor and major wave early patterning genes (59). However, there are some exceptions: (i) 1 of 10 pair-rule genes [even-skipped (eve)] was slightly down-regulated in CP190 depletion, and (ii) 2 of 8 homeotic genes had a change in expression: Scr was slightly up-regulated in BEAF-32 and down-regulated in CTCF depletions, while Ultrabithorax (Ubx) was down-regulated in CP190 depletion (table S3). ...
The boundaries of topologically associating domains (TADs) are delimited by insulators and/or active promoters; however, how they are initially established during embryogenesis remains unclear. Here, we examined this during the first hours of Drosophila embryogenesis. DNA-FISH confirms that intra-TAD pairwise proximity is established during zygotic genome activation (ZGA) but with extensive cell-to-cell heterogeneity. Most newly formed boundaries are occupied by combinations of CTCF, BEAF-32, and/or CP190. Depleting each insulator individually from chromatin revealed that TADs can still establish, although with lower insulation, with a subset of boundaries (~10%) being more dependent on specific insulators. Some weakened boundaries have aberrant gene expression due to unconstrained enhancer activity. However, the majority of misexpressed genes have no obvious direct relationship to changes in domain-boundary insulation. Deletion of an active promoter (thereby blocking transcription) at one boundary had a greater impact than deleting the insulator-bound region itself. This suggests that cross-talk between insulators and active promoters and/or transcription might reinforce domain boundary insulation during embryogenesis.
