The typical developmental trajectory and energy requirements of Shitou goose during the embryonic stage.

Low hatchability has been a persistent challenge in the goose industry. Establishing standard atlases and comprehending embryonic development patterns are essential to improving the hatching rates of goose eggs. However, comprehensive descriptions of normal atlases, embryonic development, and energy requirements in geese are lacking. In this study, a total of 120 fertile eggs from well-known large Shitou goose were incubated using 12 nesting purebred female geese. During hatching, both the temperature of the eggshells and the weight of eggs were recorded, and daily photographs of the embryos were captured to monitor their development closely. After hatching, counted the number of pores per unit area of eggshells by choosing eggs from without sperm, dead embryos, and normally hatched. Furthermore, 150 Shitou goose eggs were hatched by automatic incubator, with adjustments made based on observed normal developmental stages that incubated by female geese. The eggs were carefully opened to meticulously document embryonic morphology and create a detailed development map. Measurements were taken of the eye diameter, length of the lower beak, tarsometatarsus bone, and embryo length. Subsequently, an analysis was conducted to assess the calcium, phosphorus, crude protein, and crude fat content to study the energy requirements for embryo development. characteristics on the 7th, 15th, 23rd and 28th days of Shitou goose hatching corresponded to the 5th, 10th, 17th and 19th days of chicken egg incubation, respectively. These days were distinguished individually by "visible embryo's eye", "closure", "sealing the door", and "flashing hair". Besides, the hatch rate of the incubator reached 86.67%, and the cumulative water loss rate increased with embryo age. Notably, normally developing embryos displayed a significantly higher number of pores on the eggshell surface compared to dead embryos (P < 0.05). Additionally, embryonic body length, eyeball diameter, and lower beak length exhibited continuous growth until day 19 of incubation, while tarsometatarsus length increased steadily from days 12 to 31. Liver size measurement began on the 10th day of incubation, while both leg and chest muscles showed continuous growth from the 12th day. For energy demand, the embryo primarily relied on protein sourced from the egg yolk within the first 10 days of development. Afterward, the egg yolk provided both protein and fat for embryonic growth. In summary, this study has generated a comprehensive developmental map for Shitou goose embryos, offering valuable insights into their growth and morphological changes throughout the incubation period. This map can serve as a reference for optimizing machine incubation techniques to enhance goose egg hatching rates and provide fresh perspectives on the development of geese.

Multiple embryonic sources converge to form the pectoral girdle skeleton in zebrafish.

The morphological transformation of the pectoral/shoulder girdle is fundamental to the water-to-land transition in vertebrate evolution. Although previous studies have resolved the embryonic origins of tetrapod shoulder girdles, those of fish pectoral girdles remain uncharacterized, creating a gap in the understanding of girdle transformation mechanisms from fish to tetrapods. Here, we identify the embryonic origins of the zebrafish pectoral girdle, including the cleithrum as an ancestral girdle element lost in extant tetrapods. Our combinatorial approach of photoconversion and genetic lineage tracing demonstrates that cleithrum development combines four adjoining embryonic populations. A comparison of these pectoral girdle progenitors with extinct and extant vertebrates highlights that cleithrum loss, indispensable for neck evolution, is associated with the disappearance of its unique developmental environment at the head/trunk interface. Overall, our study establishes an embryological framework for pectoral/shoulder girdle formation and provides evolutionary trajectories from their origin in water to diversification on land.

Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud.

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early ß-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of ß-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in ß-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any ß-catenin transcriptional activity as measured by ß-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in ß-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

Expression analysis of thg1l during Xenopus laevis development.

The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.

Differential cellular stiffness across tissues that contribute to Xenopus neural tube closure.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.

Assessment of the effects of 2,4-D-dichlorophenoxyacetic acid-based herbicide exposure in eggs and embryos of golden apple snails using OCT.

This study aimed to evaluate the effects of herbicide 2, 4-D-dichlorophenoxy acetic acid on golden apple snail eggs and embryos. Additionally, the study assessed the applicability of optical coherence tomography (OCT), a non-invasive depth cross-sectional microscopic imaging technique, as a novel method, to the best of our knowledge, for studying morphological changes in golden apple snail eggs and embryos, in comparison to the conventional approach of using white light microscopy. The study revealed that the herbicide 2,4-D-dichlorophenoxy acetic acid affected the hatchery rate and morphological changes of the eggs and embryos. The lethal concentration (LC50), representing the concentration of a substance that is expected to cause death in half of the population being studied, of the golden apple eggs and embryos increased with longer exposure time and higher concentrations. The estimated median effective concentration (EC50), which denotes the concentration producing the desired effect in 50% of the exposed golden apple embryos, exhibited a similar trend of change as the LC50. When compared to the microscopic study, it was observed that OCT could be employed to investigate morphological changes of golden apple snail eggs and embryos, enabling evaluation of alterations in both 2D and 3D structures.

Cyclic muscle contractions reinforce the actomyosin motors and mediate the full elongation of C. elegans embryo.

The paramount importance of mechanical forces in morphogenesis and embryogenesis is widely recognized, but understanding the mechanism at the cellular and molecular level remains challenging. Because of its simple internal organization, Caenorhabditis elegans is a rewarding system of study. As demonstrated experimentally, after an initial period of steady elongation driven by the actomyosin network, muscle contractions operate a quasi-periodic sequence of bending, rotation, and torsion, that leads to the final fourfold size of the embryos before hatching. How actomyosin and muscles contribute to embryonic elongation is investigated here theoretically. A filamentary elastic model that converts stimuli generated by biochemical signals in the tissue into driving forces, explains embryonic deformation under actin bundles and muscle activity, and dictates mechanisms of late elongation based on the effects of energy conversion and dissipation. We quantify this dynamic transformation by stretches applied to a cylindrical structure that mimics the body shape in finite elasticity, obtaining good agreement and understanding of both wild-type and mutant embryos at all stages.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Normal table of embryonic development in the Anji salamander Hynobius amjiensis (Hynobiidae).

We established a normal embryonic development table for the Anji salamander Hynobius amjiensis, a critically endangered tailed amphibian of the family Hynobiidae with a very limited distribution in East China, following the standards set by the early developmental table of vertebrates. Put together 32 embryonic stages for the Anji salamander was defined. The total embryonic period from oviposition to hatching is approximately 30 days at 9 °C. Stages 1-16 represent early development from cleavage to neurulation. Stages 17-32 represent organogenesis documenting later developmental events such as tail, gill, and limb formation, and hatching (Stage 32). We provided a detailed description of the external morphology and color changes of the head, trunk, limbs, tail, external gills, and balancers at various stages from egg-laying to hatching. We also described several cases of abnormal embryonic development. The establishment of the embryonic development table in H. amjiensis contributes to better understanding of the ontogeny in tailed amphibians, distinguishing closely related species, and identifying abnormal embryonic amphibians.

Transfer of polarity information via diffusion of Wnt ligands in C. elegans embryos.

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how Wnt ligands, produced in the posterior half of the embryos, spread extracellularly to transmit information to distant target cells in the anterior half. With quantitative live imaging and fluorescence correlation spectroscopy, we show that Wnt ligands diffuse through the embryo over a timescale shorter than the cell cycle, in the intercellular space, and outside the tissue below the eggshell. We extracted diffusion coefficients of Wnt ligands and their receptor Frizzled and characterized their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize individual cells through a time integration of the arrival of the ligands at the target cells. The polarity established at the tissue level by a posterior Wnt source can be transferred to the cellular level. Our results support a diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10 000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Targeted design of synthetic enhancers for selected tissues in the Drosophila embryo.

Enhancers control gene expression and have crucial roles in development and homeostasis1-3. However, the targeted de novo design of enhancers with tissue-specific activities has remained challenging. Here we combine deep learning and transfer learning to design tissue-specific enhancers for five tissues in the Drosophila melanogaster embryo: the central nervous system, epidermis, gut, muscle and brain. We first train convolutional neural networks using genome-wide single-cell assay for transposase-accessible chromatin with sequencing (ATAC-seq) datasets and then fine-tune the convolutional neural networks with smaller-scale data from in vivo enhancer activity assays, yielding models with 13% to 76% positive predictive value according to cross-validation. We designed and experimentally assessed 40 synthetic enhancers (8 per tissue) in vivo, of which 31 (78%) were active and 27 (68%) functioned in the target tissue (100% for central nervous system and muscle). The strategy of combining genome-wide and small-scale functional datasets by transfer learning is generally applicable and should enable the design of tissue-, cell type- and cell state-specific enhancers in any system.

Organization of the body wall in lampreys informs the evolution of the vertebrate paired appendages.

Vertebrate paired appendages are one of the most important evolutionary novelties in vertebrates. During embryogenesis, the skeletal elements of paired appendages differentiate from the somatic mesoderm, which is a layer of lateral plate mesoderm. However, the presence of the somatic mesoderm in the common ancestor of vertebrates has been controversial. To address this problem, it is necessary but insufficient to understand the developmental process of lateral plate mesoderm formation in lamprey (jawless vertebrates) embryos. Here, I show the presence of the somatic mesoderm in lamprey (Lethenteron camtschaticum) embryos using plastic sectioning and transmission electron microscopy analysis. During the early pharyngeal stages, the somatic mesoderm transforms from the lateral plate mesoderm in the trunk region. Soon after, when the cardiac structures were morphologically distinct, the somatic mesoderm was recognized through the cardiac to more caudal regions. These findings indicated that the somatic mesoderm evolved before the emergence of paired appendages. I also discuss the developmental changes in the body wall organization in the common ancestor of vertebrates, which is likely related to the evolution of the paired appendages.

Dynamic FMR1 granule phase switch instructed by m6A modification contributes to maternal RNA decay.

Maternal RNA degradation is critical for embryogenesis and is tightly controlled by maternal RNA-binding proteins. Fragile X mental-retardation protein (FMR1) binds target mRNAs to form ribonucleoprotein (RNP) complexes/granules that control various biological processes, including early embryogenesis. However, how FMR1 recognizes target mRNAs and how FMR1-RNP granule assembly/disassembly regulates FMR1-associated mRNAs remain elusive. Here we show that Drosophila FMR1 preferentially binds mRNAs containing m6A-marked "AGACU" motif with high affinity to contributes to maternal RNA degradation. The high-affinity binding largely depends on a hydrophobic network within FMR1 KH2 domain. Importantly, this binding greatly induces FMR1 granule condensation to efficiently recruit unmodified mRNAs. The degradation of maternal mRNAs then causes granule de-condensation, allowing normal embryogenesis. Our findings reveal that sequence-specific mRNAs instruct FMR1-RNP granules to undergo a dynamic phase-switch, thus contributes to maternal mRNA decay. This mechanism may represent a general principle that regulated RNP-granules control RNA processing and normal development.

Cryopreservation of Anopheles stephensi embryos.

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15-30 min post oviposition, two incubation steps in 100% deuterated methanol at - 7 °C and - 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification.

Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.

A novel gene trap line for visualization and manipulation of erbb3b+ neural crest and glial cells in zebrafish.

Glia are a diverse and essential cell type in the vertebrate nervous system. Transgenic tools and fluorescent reporter lines are critical resources to investigate how glial subtypes develop and function. However, despite the many lines available in zebrafish, the community still lacks the ability to label all unique stages of glial development and specific subpopulations of cells. To address this issue, we screened zebrafish gene and enhancer trap lines to find a novel reporter for peripheral glial subtypes. From these, we generated the gSAIzGFFD37A transgenic line that expresses GFP in neural crest cells and central and peripheral glia. We found that the gene trap construct is located within an intron of erbb3b, a gene essential for glial development. Additionally, we confirmed that GFP+ â€‹cells express erbb3b along with sox10, a known glial marker. From our screen, we have identified the gSAIzGFFD37A line as a novel and powerful tool for studying glia in the developing zebrafish, as well as a new resource to manipulate erbb3b+ â€‹cells.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-ß-induced tumor-associated fibrosis.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-ß-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

Germline development during embryogenesis of the larvacean, Oikopleura dioica.

Germ cells develop into eggs and sperms and represent a lineage that survives through multiple generations. Germ cell specification during embryogenesis proceeds through one of two basic modes: either the cell-autonomous mode or the inductive mode. In the cell-autonomous mode, specification of germ cell fate involves asymmetric partitioning of the specialized maternal cytoplasm, known as the germplasm. Oikopleura dioica is a larvacean (class Appendicularia) and a chordate. It is regarded as a promising animal model for studying chordate development because of its short life cycle (5 days) and small genome size (∼60 â€‹Mb). We show that their embryos possess germplasm, as observed in ascidians (class Ascidiacea). The vegetal cytoplasm shifted towards the future posterior pole before the first cleavage occurred. A bilateral pair of primordial germ cells (PGC, B11 â€‹cells) was formed at the posterior pole at the 32-cell stage through two rounds of unequal cleavage. These B11 â€‹cells did not undergo further division before hatching of the tadpole-shaped larvae. The centrosome-attracting body (CAB) is a subcellular structure that contains the germplasm and plays crucial roles in germ cell development in ascidians. The presence of CAB with germplasm was observed in the germline lineage cells of larvaceans via electron microscopy and using extracted embryos. The CAB appeared at the 8-cell stage and persisted until the middle stage of embryogenesis. The antigen for the phosphorylated histone 3 antibody was localized to the CAB and persisted in the PGC until hatching after the CAB disappeared. Maternal snail mRNA, which encodes a transcription factor, was co-localized with the antigen for the H3S28p antibody. Furthermore, we found a novel PGC-specific subcellular structure that we call the germ body (GB). This study thus highlights the conserved and non-conserved features of germline development between ascidians and larvaceans. The rapid development and short life cycle (five days) of O. dioica would open the way to genetically analyze germ cell development in the future.

Transcription and splicing dynamics during early Drosophila development.

Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.