Blastoderm Stage Research Articles

Real-time visualisation of developing chick embryos cultured in transparent plastic films from the blastoderm stage until hatching

Real-time visualisation of chick embryo development occurring inside an opaque eggshell is a major goal of developmental biology. This goal was partially achieved when 3-day-old embryos recovered from fertilised shelled eggs, preincubated in a conventional incubator, hatched after the egg contents had been transferred into shell-less embryo culture systems constructed of transparent plastic film placed in a plastic cup. However, the hatchable shell-less embryo culture systems described thus far cannot be used to visualise the dynamic morphological changes associated with the emergence of major organ systems during the first 3 days of embryo development, as the blastoderm area needs to be covered with an opaque membrane. Here, we present the development of a chick embryo culture system that enables omnidirectional real-time visualisation of the developing embryo, beginning from the newly laid blastoderm stage embryo until hatching.

Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example andpresents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Evolution of patterning.

Developing tissues are patterned in space and time; this enables them to differentiate their cell types and form complex structures to support different body plans. Although space and time are two independent entities, there are many examples of spatial patterns that originate from temporal ones. The most prominent example is the expression of the genes hunchback, Krüppel, pdm, and castor, which are expressed temporally in the neural stem cells of the Drosophila ventral nerve cord and spatially along the anteroposterior axis of the blastoderm stage embryo. In this Viewpoint, we investigate the relationship between space and time in specific examples of spatial and temporal patterns with the aim of gaining insight into the evolutionary history of patterning.

The ftz upstream element drives late ftz stripes but is not required for regulation of ftz target genes

The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. Here, we asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS7S homozygotes, ftz stripes appear in the same order as wildtype and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz striped expression, with all stripes decaying earlier than wildtype in ftzΔUPS7S homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. We propose that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, our results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development.

Rapid egg cooling rate after oviposition influences the embryonic development, hatchability, and hatch time of young and old broiler hatching eggs

This study investigated the effect of the egg cooling profile after oviposition on blastoderm development, embryonic mortality, hatchability, and hatch time of broiler hatching eggs from young and old breeder flocks. Hatching eggs were obtained from commercial Ross 308 broiler breeders at 28 wk (young) and 64 wk (old) of age. A total of 3,150 eggs laid within a 15-min period were collected and randomly assigned to 2 temperature-controlled chambers in both flocks. The eggshell temperature (EST) was cooled to 24°C either within 6 h (control) or 45 min (rapid). After the EST reached 16°C in the chambers in all groups, eggs were transported to the commercial hatchery. Eggs were stored for 6 d at 16°C and 75% relative humidity. The development of the blastoderm in sampled eggs (25 embryos in each batch) was determined immediately after egg collection and before transport to the hatchery (after cooling) on a farm in each flock. At each flock age, there were 5 replicate trays of 150 eggs per egg cooling treatment set in a single commercial incubator. The results showed that the embryonic developmental stage was retarded by rapid cooling and by the younger flock. A flock age×cooling rate interaction was observed for fertile hatchability and early and late embryonic mortality (P < 0.001). In the young flock eggs, the fertile hatchability was significantly lower in the rapid than in the control cooling treatment (88.7 vs. 92.8%) due to higher early and late embryonic mortality, whereas rapid cooling reduced early embryonic mortality (P < 0.01) and numerically increased the fertile hatchability (88.7 vs. 87.2%) in the old flock eggs. Hatch time was affected by the cooling treatment. The average hatch time was delayed by 3 h by rapid cooling (486.2 vs. 489.2 h) after oviposition compared with the control. This study showed that cooling the EST to 24°C within 45 min (rapid cooling) compared to 6 h (control) after laying retarded the blastoderm developmental stage and hatch time of eggs from both young and old broiler breeder flocks. This was apparently detrimental for the young flock as indicated by the higher early and late embryonic mortality but beneficial for the old flock due to the lower early embryonic mortality. The differences in hatchability between young and old flock eggs resulting from a rapid cooling rate might depend on the differences in embryonic development at oviposition.

Shell-less culture system for chick embryos from the blastoderm stage to hatching.

Since 2014, methods have been described to hatch chick embryos from shell-less culture after egg contents are first incubated within shells for 55-70 h. The present report describes for the first time a shell-less culture system for chick embryos from the blastoderm stage to hatching. For the first 69-70 h, egg contents suspended in polymethylpentene kitchen wrap (F.O.R. Wrap, Riken Fabro, Tokyo, Japan) supported in 6.35 or 6.67 cm inside diameter tripods and covered with a disc of immobilized Milli-Wrap, were rotated back and forth through 90°at 16 or 22 cycles per minute (CPM). Subsequently, the Milli-Wrap disc was removed and culture tripods were transferred to environmental chambers, which were rocked ±20° through incubation day 8.5 (E8.5). From E9, environmental chambers were maintained in the horizontal position through to hatching with controlled O2 and CO2 . To provide supplemental calcium, an aqueous solution containing 100 mg/mL of calcium l-lactate hydrate was injected through the plastic wrap into the albumen at E9 (2.5 mL) and at E13 (1.0 mL) or E15 (1.0 mL). After incubation for 69-70 h at 16 or 22 CPM, 80%-83% of previously unincubated egg contents yielded apparently normal embryos. Hatch rate of normal embryos resulting from turntable incubation at 16 or 22 CPM was approximately 43%. Of note, egg contents remained in the same culture tripod from blastoderm stage to hatching. This technique may find use as an educational tool and in basic investigations of early embryogenesis, teratogenesis, and gene transfer experiments.

A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development.

The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA “in cubo”, followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the “in cubo” electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Quantification reveals early dynamics in Drosophila maternal gradients.

The Bicoid (Bcd) protein is a primary determinant of early anterior-posterior (AP) axis specification in Drosophila embryogenesis. This morphogen is spatially distributed in an anterior-high gradient, and affects particular AP cell fates in a concentration-dependent manner. The early distribution and dynamics of the bicoid (bcd) mRNA, the source for the Bcd protein gradient, is not well understood, leaving a number of open questions for how Bcd positional information develops and is regulated. Confocal microscope images of whole early embryos, stained for bcd mRNA or the Staufen (Stau) protein involved in its transport, were processed to extract quantitative AP intensity profiles at two depths (apical-under the embryo surface but above the nuclear layer; and basal-below the nuclei). Each profile was quantified by a two- (or three-) exponential equation. The parameters of these equations were used to analyze the early developmental dynamics of bcd. Analysis of 1D profiles was compared with 2D intensity surfaces from the same images. This approach reveals strong early changes in bcd and Stau, which appear to be coordinated. We can unambiguously discriminate three stages in early development using the exponential parameters: pre-blastoderm (1-9 cleavage cycle, cc), syncytial blastoderm (10-13 cc) and cellularization (from 14A cc). Key features which differ in this period are how fast the first exponential (anterior component) of the apical profile drops with distance and whether it is higher or lower than the basal first exponential. We can further discriminate early and late embryos within the pre-blastoderm stage, depending on how quickly the anterior exponential drops. This relates to the posterior-wards spread of bcd in the first hour of development. Both bcd and Stau show several redistributions in the head cytoplasm, quite probably related to nuclear activity: first shifting inwards towards the core plasm, forming either protrusions (early pre-blastoderm) or round aggregations (early nuclear cleavage cycles, cc, 13 and 14), then moving to the embryo surface and spreading posteriorly. These movements are seen both with the 2D surface study and the 1D profile analysis. The continued spreading of bcd can be tracked from the time of nuclear layer formation (later pre-blastoderm) to the later syncytial blastoderm stages by the progressive loss of steepness of the apical anterior exponential (for both bcd and Stau). Finally, at the beginning of cc14 (cellularization stage) we see a distinctive flip from the basal anterior gradient being higher to the apical gradient being higher (for both bcd and Stau). Quantitative analysis reveals substantial (and correlated) bcd and Stau redistributions during early development, supporting that the distribution and dynamics of bcd mRNA are key factors in the formation and maintenance of the Bcd protein morphogenetic gradient. This analysis reveals the complex and dynamic nature of bcd redistribution, particularly in the head cytoplasm. These resemble observations in oogenesis; their role and significance have yet to be clarified. The observed co-localization during redistribution of bcd and Stau may indicate the involvement of active transport.

Dynamic transcriptome analysis of Bombyx mori embryonic development.

Bombyx mori has been extensively studied but the gene expression control of its embryonic development is unclear. In this study, we performed transcriptome profiling of six stages of B. mori embryonic development using RNA sequencing (RNA-seq). A total of 12894 transcripts were obtained from the embryos. Of these, 12456 transcripts were shared among the six stages, namely, fertilized egg, blastoderm, germ-band, organogenesis, reversal period, and youth period stages. There were 111, 48, 41, 54, 77, and 107 transcripts specifically expressed during the six stages, respectively. By analyzing weighted gene correlation networks and differently expressed genes, we found that during embryonic development, many genes related to DNA replication, transcription, protein synthesis, and epigenetic modifications were upregulated in the early embryos. Genes of cuticle proteins, chitin synthesis-related proteins, and neuropeptides were more abundant in the late embryos. Although pathways of juvenile hormone and the ecdysteroid 20-hydroxyecdysone, and transcription factors were expressed throughout the embryonic development stages, more regulatory pathways were highly expressed around the organogenesis stage, suggesting more gene expression for organogenesis. The results of RNA-seq were confirmed by quantitative real-time polymerase chain reaction of 16 genes of different pathways. Nucleic acid methylation and seven sites in histone H3 modifications were confirmed by dot blot and western blot. This study increases the understanding of the molecular mechanisms of the embryonic developmental process and information on the regulation of B. mori development.

Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Identification and characterization of four Drosophila suzukii cellularization genes and their promoters

BackgroundThe spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product.ResultsHere, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5′ flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells.ConclusionsThe similarities in the gene coding and 5′ flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

Transcriptome analysis revealed misregulated gene expression in blastoderms of interspecific chicken and Japanese quail F1 hybrids.

Hybrid incompatibility, such as sterility and inviability, prevents gene flow between closely-related populations as a reproductive isolation barrier. F1 hybrids between chickens and Japanese quail (hereafter, referred to as quail), exhibit a high frequency of developmental arrest at the preprimitive streak stage. To investigate the molecular basis of the developmental arrest at the preprimitive streak stage in chicken-quail F1 hybrid embryos, we investigated chromosomal abnormalities in the hybrid embryos using molecular cytogenetic analysis. In addition, we quantified gene expression in parental species and chicken- and quail-derived allele-specific expression in the hybrids at the early blastoderm and preprimitive streak stages by mRNA sequencing. Subsequently, we compared the directions of change in gene expression, including upregulation, downregulation, or no change, from the early blastoderm stage to the preprimitive streak stage between parental species and their hybrids. Chromosome analysis revealed that the cells of the hybrid embryos contained a fifty-fifty mixture of parental chromosomes, and numerical chromosomal abnormalities were hardly observed in the hybrid cells. Gene expression analysis revealed that a part of the genes that were upregulated from the early blastoderm stage to the preprimitive streak stage in both parental species exhibited no upregulation of both chicken- and quail-derived alleles in the hybrids. GO term enrichment analysis revealed that these misregulated genes are involved in various biological processes, including ribosome-mediated protein synthesis and cell proliferation. Furthermore, the misregulated genes included genes involved in early embryonic development, such as primitive streak formation and gastrulation. These results suggest that numerical chromosomal abnormalities due to a segregation failure does not cause the lethality of chicken-quail hybrid embryos, and that the downregulated expression of the genes that are involved in various biological processes, including translation and primitive streak formation, mainly causes the developmental arrest at the preprimitive streak stage in the hybrids.

The multiple roles of caudal in early development of the milkweed bug Oncopeltus fasciatus

The homeobox transcription factor Caudal has conserved roles in all Bilateria in defining the posterior pole and in controlling posterior elongation. These roles are seemingly similar and are difficult to disentangle. We have carried out a detailed analysis of the expression, function and interactions of the caudal ortholog of the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect with a conservative early development process, in order to understand its different functions throughout development. In Oncopeltus, caudal is not maternally deposited, but has a sequence of roles in the posterior of the embryos throughout early development. It defines and maintains a posterior-anterior gradient in the blastoderm and modulates the activity of segmentation genes in simultaneous segmentation during the blastoderm stage. It later defines the invagination site and the posterior segment addition zone (SAZ) in the germband. It maintains the posterior SAZ cells in an undifferentiated proliferative state, while promoting dynamic expression of segmentation genes in the anterior SAZ. We show that rather than being a simple posterior determinant, Caudal is involved in several distinct regulatory networks, each with a distinct developmental role.

Two pair-rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo.

While many developmentally relevant enhancers act in a modular fashion, there is growing evidence for nonadditive interactions between distinct cis-regulatory enhancers. We investigated if nonautonomous enhancer interactions underlie transcription regulation of the Drosophila segment polarity gene, wingless. We identified two wg enhancers active at the blastoderm stage: wg 3613u, located from -3.6 to -1.3 kb upstream of the wg transcription start site (TSS) and 3046d, located in intron two of the wg gene, from 3.0 to 4.6 kb downstream of the TSS. Genetic experiments confirm that Even Skipped (Eve), Fushi-tarazu (Ftz), Runt, Odd-paired (Opa), Odd-skipped (Odd), and Paired (Prd) contribute to spatially regulated wg expression. Interestingly, there are enhancer specific differences in response to the gain or loss of function of pair-rule gene activity. Although each element recapitulates aspects of wg expression, a composite reporter containing both enhancers more faithfully recapitulates wg regulation than would be predicted from the sum of their individual responses. These results suggest that the regulation of wg by pair-rule genes involves nonadditive interactions between distinct cis-regulatory enhancers.

PRDM14 and BLIMP1 control the development of chicken primordial germ cells

The differentiation of primordial germ cells (PGCs) is a fundamental step in development. PR domain-containing protein 14 (PRDM14) and B lymphocyte-induced maturation protein 1 (BLIMP1) play pivotal roles in mouse PGC specification. In the present study, we assessed the roles of chicken orthologs of PRDM14 and BLIMP1 in PGC development. PRDM14 and BLIMP1 were expressed in blastodermal cells and PGCs. The in vivo knockdown of PRDM14 or BLIMP1 by introducing a replication-competent retroviral vector expressing shRNAs to the blastodermal stage of embryos reduced the number of SSEA-1 or chicken vasa homologue-positive PGCs on day 5.5–6.5. Since the inhibition of Activin receptor-like kinase 4/5/7 in cultured PGCs reduced the expression of PRDM14, BLIMP1, and NANOG, and that of MEK inhibited PRDM14 expression, the expression of these genes seems to be controlled by Activin A and FGF2 signaling. Overall, PRDM14, BLIMP1, and NANOG seem to be involved in the self-renewal of PGCs in cultured PGCs and embryos.

Physically-Inspired Gaussian Process Models for Post-Transcriptional Regulation in Drosophila.

The regulatory process of Drosophila is thoroughly studied for understanding a great variety of biological principles. While pattern-forming gene networks are analyzed in the transcription step, post-transcriptional events (e.g., translation, protein processing) play an important role in establishing protein expression patterns and levels. Since the post-transcriptional regulation of Drosophila depends on spatiotemporal interactions between mRNAs and gap proteins, proper physically-inspired stochastic models are required to study the link between both quantities. Previous research attempts have shown that using Gaussian processes (GPs) and differential equations lead to promising predictions when analyzing regulatory networks. Here, we aim at further investigating two types of physically-inspired GP models based on a reaction-diffusion equation where the main difference lies in where the prior is placed. While one of them has been studied previously using protein data only, the other is novel and yields a simple approach requiring only the differentiation of kernel functions. In contrast to other stochastic frameworks, discretizing the spatial space is not required here. Both GP models are tested under different conditions depending on the availability of gap gene mRNA expression data. Finally, their performances are assessed on a high-resolution dataset describing the blastoderm stage of the early embryo of Drosophila melanogaster.

A Role for buttonhead in the Early Head and Trunk Development in the Beetle Tribolium castaneum.

Thehead gap gene buttonhead (btd) is required for the patterning of head segments in the early Drosophila embryo. Mutant phenotypes of btd display a gap-like phenotype in which antennal, intercalary, mandibular and the anterior portion of the maxillary segmentsare eliminated. In agreement with the phenotypes, btd is expressed in a stripe covering the head segments at the blastoderm stage. During the early phase of the germband extension, btd is expressed in stripes with single segmental periodicity, which is required for the formation of the peripheral nervous system. In contrast to the key role of btd in Drosophila embryonic development, it has been suggested that Tribolium ortholog of btd (Tc-btd) is dispensable for embryonic head development. In order for better understanding of the requirement of Tc-btd in the early Tribolium embryo, we re-analyzed the expression patterns and functions of Tc-btd during embryonic segmentation. Tc-btd is expressed in segmental stripes at the stages of blastoderm and germband elongation. Up to 28.3% of embryos in which Tc-btd is knocked down displays the loss of antennal, mandibular and the pregnathal regions in the head, with abdominal segments being disrupted in the trunk. Our findings suggest that Tc-btd is required for the head and trunk development in the early Tribolium embryo.