Formation In Embryo Research Articles

Interplay between Lefty and Nodal signaling is essential for the organizer and axial formation in amphioxus embryos

The organizer is an essential signaling center required for axial formation during vertebrate embryonic development. In the basal chordate amphioxus, the dorsal blastopore lip of the gastrula has been proposed to be homologous to the vertebrate organizer. Lefty is one of the first genes to be expressed in the organizer. The present results show that Lefty overexpression abolishes the organizer; the embryos were severely ventralized and posteriorized, and failed to develop anterior and dorsal structures. In Lefty knockouts the organizer is enlarged, and anterior and dorsal structures are expanded. Different from Lefty morphants in vertebrates, amphioxus Lefty mutants also exhibited left-right defects. Inhibition of Nodal with SB505124 partially rescued the effects of Lefty loss-of-function on morphology. In addition, while SB505124 treatment blocked Lefty expression in the cleavage stages of amphioxus embryos, activation of Nodal signaling with Activin protein induced ectopic Lefty expression at these stages. These results show that the interplay between Lefty and Nodal signaling plays an essential role in the specification of the amphioxus organizer and axes.

Electrical stimulation of developmental forces reveals the mechanism of limb formation in vertebrate embryos.

Current knowledge on limbs development lacks a physical description of the forces leading to formation of the limbs precursors or "buds". Earlier stages of development are driven by large scale morphogenetic movements, such as dipolar vortical flows and mechanical buckling, pulled by rings of cells. It is a natural hypothesis that similar phenomena occur during limb formation. However it is difficult to experiment on the developmental forces, in such a complex dynamic system. Here, we report a physical study of hindlimb bud formation in the chicken embryo. We use electrical stimulation to enhance the physical forces present in the tissue, prior to limb bud formation. By triggering the physical forces in a rapid and amplified pattern, we reveal the mechanism of formation of the hindlimbs: the early presumptive embryonic territory is composed of a set of rings encased like Russian dolls. Each ring constricts in an excitable pattern of force, and the limb buds are generated by folding at a pre-existing boundary between two rings, forming the dorsal and ventral ectoderms. The amniotic sac buckles at another boundary. Physiologically, the actuator of the excitable force is the tail bud pushing posteriorly along the median axis. The developmental dynamics suggests how animals may evolve by modification of the magnitude of these forces, within a common broken symmetry. On a practical level, localized electrical stimulation of morphogenetic forces opens the way to in vivo electrical engineering of tissues.

A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos

BackgroundThe segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively.ResultsWe show that SOX17 starts to be expressed in 16–32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor.ConclusionsThis study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 μM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

In vitro production of canine blastocysts

Though blastocyst production in vitro has been successful in several animal species, a culture system to produce viable and normal canine blastocysts in vitro remains to be established. In this study, we report the development of an in vitro culture system for canine preimplantation embryos produced via parthenogenetic activation (PA) and somatic cell nucleus transfer (SCNT). Our results show that the medium developed by us, named “Qingdao Agricultural University's (QAU)-4 medium”, successfully breaks the developmental arrest observed at the eight-cell stage in canine embryos grown in other culture systems. The blastocysts produced in QAU-4 displayed normal blastocyst structures, including a clear inner cell mass and blastocyst cavity. We also found that blastocyst formation in PA embryos cultured in QAU-4 medium was quite high, though this was not so in the case of SCNT embryos. However, supplementation of QAU-4 medium with 100 nM of scriptaid caused a sharp increase in blastocyst formation in SCNT embryos. After culture, hatched blastocysts were also observed to successfully adhere to collagen-coated dishes, where further growth and differentiation occurred. To our knowledge, this is the first in vitro canine preimplantation embryo culture system that can successfully produce canine blastocysts.

Vesicular swelling in the cervical region with lymph sac formation in human embryos.

Vesicular swelling in the cervical region (VSC) is occasionally observed among human embryos around Carnegie stage (CS) 21. However, its mechanism and significance in fetal development are unclear. The present study aimed to analyze the relation of development of VSC with jugular lymph sac (JLS) formation. Serial histological sections that were digitalized from 14 embryos at CS20 and CS21 stored at the Kyoto Collection were used for the analysis. Subcutaneous edema and enlargement of the subarachnoid space were found to cause VSC. No obvious abnormalities in cranial regions that may be related to the VSC were detected on histological sections. Three-dimensional reconstructions revealed the following: (a) the JLS was located bilaterally at the levels between the first and fourth cervical vertebrae; (b) the JLS was pyramidal in shape; and (c) no severe deformity and/or malformation was found in all samples. The JLS was not connected to the subcutaneous tissue and subarachnoid space in all samples. The mean volume of the JLS increased nine-times from CS20 (0.02 mm3 in VSC [-] group) to CS21 (0.18 mm3 in VSC [-] group). The mean volume of the JLS was comparable between the VSC [-] and VSC (+) groups at both CS20 and CS21. A moderate correlation was observed between VSCd and the mean volume of the JLS in both groups at CS20 (R2 = 0.75) and CS21 (R2 = 0.56). In conclusion, the dynamics of the lymphatic system at the cervical region may contribute to VSC observed around CS21. © 2019 Japanese Teratology Society.

Macrophages Fertilize the Soil to Promote Hematopoietic Cell Growth

Inflammatory signals support the birth of hematopoietic stem cells in zebrafish embryos, but their cellular source in mammals is not known. In this issue, Mariani etal. (2019) report that macrophages are a primary source of pro-inflammatory signals that promote blood cell formation in mammalian embryos.

Understanding Pattern Formation in Embryos: Experiment, Theory, and Simulation

Twenty-five years ago, Lewis Wolpert, the eminent developmental biologist, asked the question, "Do We Understand Development?" He concluded that such rapid progress had been made in the preceding two decades that "It is not unreasonable to think that enough will eventually be known to program a computer and simulate some aspects of development." This prediction has been fulfilled, at least partially, with data-driven simulations of several different developmental processes being developed in the intervening years. Nevertheless, the question remains of whether we "understand" development and if simulations are sufficient to provide an explanation of development. While in silico replications and models are undoubtedly an important tool in the investigation and dissection of developmental processes, which complement traditional experimental methods, these need to be supplemented by theory that identifies principles and provides coherent explanations. Here, I use the example of pattern formation in the vertebrate neural tube to illustrate this idea.

Pro-angiogenic activity of isoliquiritin on HUVECs in vitro and zebrafish in vivo through Raf/MEK signaling pathway

AimsLiquorice is a widely used herbal medicine for treating various diseases native to southern Europe and parts of Asia. Isoliquiritin (ISL), a licorice root-derived flavonoid, has been reported to exhibit antioxidant, anti-inflammatory, anti-genotoxic activity and anti-depression activities. This study was aimed to explore the pro-angiogenic activity of ISL and explicate the underlying mechanism. Main methodsIn vitro, ISL-treated human umbilical vein endothelial cells (HUVECs) were analyzed for cell viability, cell migration and tube formation. In vivo, pro-angiogenic effects were evaluated for the intersegmental vessels (ISVs) formation in transgenic zebrafish embryos [Tg(fli-1: EGFP)]. Furthermore, a blocking assay with eight pathways-specific kinase inhibitors were also used to determine the potential pro-angiogenic mechanism of ISL. Key findingsISL counteracted tyrosine kinase inhibitor II (VRI)-induced endothelial cell apoptosis and promoted cell migration and tube formation in HUVECs. ISL markedly rescued ISVs loss induced by VRI in zebrafish embryos, probably by activating vascular endothelial growth factor receptor-2 (VEGFR-2), phosphoinositide 3-kinase (PI3K), Raf and mitogen-activated protein kinase (MEK)-dependent signaling pathways. SignificanceOur study first discovered and confirmed the pro-angiogenic activity of ISL both in HUVECs and zebrafish. Thus, ISL could be developed as a potential therapeutic agent by the role of pro-angiogenic activity for the treatment of cardiovascular diseases, cerebrovascular diseases and other vascular diseases.

Stearoyl-coenzyme A desaturase 1 is required for lipid droplet formation in pig embryo.

Lipid droplets (LD) provide a source of energy, and their importance during embryogenesis has been increasingly recognized. In particular, pig embryos have larger amounts of intercellular lipid bilayers than other mammalian species, suggesting that porcine embryos are more dependent on lipid metabolic pathways. The objective of the present study was to detect the effect of stearoyl-coenzyme A desaturase 1 (SCD1) on LD formation and to associate these effects with the mRNA abundance of LD formation-related genes (SREBP, ARF1, COPG2, PLD1 and ERK2) in in vitro-produced porcine embryos. To determine the effect of SCD1 on LD formation and related genes, we examined the effects of SCD1 inhibition using CAY10566 (an SCD1 inhibitor, 50 μM) on parthenogenetic embryos. SCD1 inhibition downregulated the mRNA levels of LD formation-related genes and embryo development. Our results revealed that SCD1 functions in the regulation of LD formation via phospholipid formation and embryo development. In addition, we treated parthenogenetic embryos with oleic acid (100 μM), which led to a significant increase in the blastocyst formation rate, LD size and number compared to controls. Remarkably, the adverse effects of the SCD1 inhibitor could be counteracted by oleic acid. These data suggest that porcine embryos can use exogenous oleic acid as a metabolic energy source.

Dynamin Binding Protein Is Required for Xenopus laevis Kidney Development.

The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and water absorption and waste excretion. The embryonic kidney of Xenopus laevis consists of a single functional nephron composed of regions that are analogous to those found in the human nephron, making it a simple model for the study of nephrogenesis. The exocyst complex, which traffics proteins to the cell membrane in vesicles via CDC42, is essential for normal kidney development. Here, we show that the CDC42-GEF, dynamin binding protein (Dnmbp/Tuba), is essential for nephrogenesis in Xenopus. dnmbp is expressed in Xenopus embryo kidneys during development, and knockdown of Dnmbp using two separate morpholino antisense oligonucleotides results in reduced expression of late pronephric markers, whereas the expression of early markers of nephrogenesis remains unchanged. A greater reduction in expression of markers of differentiated distal and connecting tubules was seen in comparison to proximal tubule markers, indicating that Dnmbp reduction may have a greater impact on distal and connecting tubule differentiation. Additionally, Dnmbp reduction results in glomus and ciliary defects. dnmbp knockout using CRISPR results in a similar reduction of late markers of pronephric tubulogenesis and also results in edema formation in later stage embryos. Overexpression of dnmbp in the kidney also resulted in disrupted pronephric tubules, suggesting that dnmbp levels in the developing kidney are tightly regulated, with either increased or decreased levels leading to developmental defects. Together, these data suggest that Dnmbp is required for nephrogenesis.

Metabolism responses to silver nanoparticles stresses during zebrafish embryogenesis

Silver nanoparticles (AgNPs) are widely used in medical, optoelectronic, new energy and other related fields on account of their broad-spectrum antibacterial properties. However, their biological effects on aquatic organism embryogenesis as well as potential molecular characters and mechanisms are still poorly understood. In this study, microarray analysis unveiled the altered expression of metabolic genes in AgNPs-stressed zebrafish embryos at 24 hpf (hour post fertilization). The enrichment in pathways for the down-regulated differentially expressed genes (DEGs) indicated that genes in the metabolic pathways, such as glycolysis/gluconeogenesis, tyrosine metabolism, and fatty acid metabolism, were over-enriched for the gene lists with differential expression in both AgNPs-exposed and AgNPs-injected embryos. It was found that the down-regulated expression of the genes in glycolysis and tyrosine metabolic pathways in AgNPs-stressed embryos at 24 hpf was followed by a compensatory increased expression in embryos at 72 hpf. AgNPs might influence transcriptional expression of the glycolysis and tyrosine genes differentially through their particle and ion portions separately. Additionally, decreased hexokinase & succinate de-hydrogenase activities in glycolysis pathway and high level of pyruvate were observed in the AgNPs stressed embryos, which was similar to the metabolic molecular characters observed in progressive muscular dystrophy patients. Moreover, the altered glycolysis metabolic activities were assumed to influence the muscle fibrils maintenance via regulating the concentration of glycolysis metabolites rather than cell apoptosis and proliferation in trunk muscle cells. Furthermore, the altered tyrosine metabolic activities might be another contributor to touch response defects parallel to the defective neural circuit formation in AgNPs-stressed embryos.

Rescue of hematopoietic stem/progenitor cells formation in plcg1 zebrafish mutant

Hematopoietic stem/progenitor cells (HSPC) in zebrafish emerge from the aortic hemogenic endothelium (HE) and migrate towards the caudal hematopoietic tissue (CHT), where they expand and differentiate during definitive hematopoiesis. Phospholipase C gamma 1 (Plcγ1) has been implicated for hematopoiesis in vivo and in vitro and is also required to drive arterial and HSPC formation. Genetic mutation in plcg1−/− (y10 allele) completely disrupts the aortic blood flow, specification of arterial fate, and HSPC formation in zebrafish embryos. We previously demonstrated that ginger treatment promoted definitive hematopoiesis via Bmp signaling. In this paper, we focus on HSPC development in plcg1−/− mutants and show that ginger/10-gingerol (10-G) can rescue the expression of arterial and HSPC markers in the HE and CHT in plcg1−/− mutant embryos. We demonstrate that ginger can induce scl/runx1 expression, and that rescued HE fate is dependent on Bmp and Notch. Bmp and Notch are known to regulate nitric oxide (NO) production and NO can induce hematopoietic stem cell fate. We show that ginger produces a robust up-regulation of NO. Taken together, we suggest in this paper that Bmp, Notch and NO are potential players that mediate the effect of ginger/10-G for rescuing the genetic defects in blood vessel specification and HSPC formation in plcg1−/− mutants. Understanding the molecular mechanisms of HSPC development in vivo is critical for understanding HSPC expansion, which will have a positive impact in regenerative medicine.

Fabrication of a nanoparticle-containing 3D porous bone scaffold with proangiogenic and antibacterial properties

3D porous scaffolds based on agarose and nanocrystalline apatite, two structural components that act as a temporary mineralized extracellular matrix, were prepared by the GELPOR3D method. This shaping technology allows the introduction of thermally-labile molecules within the scaffolds during the fabrication procedure. An angiogenic protein, Vascular Endothelial Growth Factor, and an antibiotic, cephalexin, loaded in mesoporous silica nanoparticles, were included to design multifunctional scaffolds for bone reconstruction. The dual release of both molecules showed a marked increase in the number of blood vessels on embryonic day 14 in chicken embryos grown ex ovo, while, at the same time providing an antibiotic local concentration capable of inhibiting Staphylococcus aureus bacterial growth. In this sense, different release patterns, monitored by UV-spectroscopy, could be tailored as a function of the cephalexin loading strategy, either releasing all the loaded cephalexin in the first 4 h or less than 50% after 24 h. The scaffold surface was characterized by a high hydrophilicity, with contact angles between 50° and 63°, which enabled the adhesion and proliferation of preosteoblastic cells. Statement of SignificanceThe localized delivery of bioactive molecules has attracted significant attention due to the potential for dose reduction as well as reduced side effects compared to systemic delivery. In this article multifunctional 3D porous scaffolds with a designed porosity have been fabricated. The method also enables the controlled loading of an antibiotic drug and an angiogenic protein into the scaffold. These scaffolds, whose composition resembles the extracellular matrix are suitable for the adhesion of preosteoblast cells, exhibit a sustained cephalexin delivery adequate for inhibiting bacterial growth as well as release the proangiogenic molecule which induces blood vessel formation in chicken embryos grown ex ovo.

Impaired intermediate formation in mouse embryos expressing reduced levels of Tbx6.

Intermediate mesoderm (IM) is the strip of tissue lying between the paraxial mesoderm (PAM) and the lateral plate mesoderm that gives rise to the kidneys and gonads. Chick fate mapping studies suggest that IM is specified shortly after cells leave the primitive streak and that these cells do not require external signals to express IM-specific genes. Surgical manipulations of the chick embryo, however, revealed that PAM-specific signals are required for IM differentiation into pronephros-the first kidney. Here, we use a genetic approach in mice to examine the dependency of IM on proper PAM formation. In Tbx6 null mutant embryos, which form 7-9 improperly patterned anterior somites, IM formation is severely compromised, while in Tbx6 hypomorphic embryos, where somites form but are improperly patterned along the axis, the impact to IM formation is lessened. These results suggest that IM and its derivatives, the kidneys and the gonads, are directly or indirectly dependent on proper PAM formation. This has implications for humans harboring Tbx6 mutations which are known to have somite-derived defects including congenital scoliosis.

Comparison of the effects of levetiracetam and valproic acid on neural tube defect formation in the chick embryo: an experimental study

Comparison of the effects of levetiracetam and valproic acid on neural tube defect formation in the chick embryo: an experimental study

The necessity of TEAD4 for early development and gene expression involved in differentiation in porcine embryos

TEA domain family transcription factor 4 (Tead4) is known to be important for the trophectoderm (TE) segregation in murine embryos. However, the role of TEAD4 in early development of porcine embryos is still unknown. We examined TEAD4 expression patterns and attempted to determine the functions of TEAD4 during porcine preimplantation development using RNA interference. TEAD4 mRNA was upregulated from the 2–4-cell to 8–16-cell stages and then decreased to the blastocyst stage. Nuclear localization of TEAD4 protein was detected at the 16-cell stage, as well as at subsequent developmental stages. In porcine embryos injected with TEAD4 siRNA, transformation from morula to blastocyst was inhibited. Although TEAD4 downregulation did not affect the expression levels of POU class 5 homeobox 1 (OCT-4), transcription of SRY-related HMG-box gene 2 (SOX2) was detected at high level in TEAD4-downregulated embryos. It is possible that TEAD4 contributes to blastocyst formation in porcine embryos through downregulation of SOX2 expression. Collectively, our results indicate that TEAD4 is an important factor for the preimplantation development of porcine embryos.

Binding of Resveratrol to Vascular Endothelial Growth Factor Suppresses Angiogenesis by Inhibiting the Receptor Signaling

Resveratrol is a polyphenol commonly found in plants and food health products, such as grape and red wine, and was identified for its binding to vascular endothelial growth factor (VEGF) by using HerboChips screening. The binding, therefore, resulted in alterations of VEGF binding to its receptor and revealed the roles of VEGF in angiogenesis. Several lines of evidence gave support to the inhibitory activities of resveratrol in VEGF-triggered angiogenesis. In human umbilical vein endothelial cells (HUVECs), compared with a VEGF-induced group, resveratrol, at a high concentration, suppressed VEGF-mediated endothelial cell proliferation, cell migration, cell invasion, and tube formation by 80 ± 9.01%, 140 ± 3.78%, 110 ± 7.51%, and 120 ± 10.26%, respectively. Moreover, resveratrol inhibited the subintestinal vessel formation in zebrafish embryo. In signaling cascades, application of resveratrol in HUVECs reduced the VEGF-triggered VEGF receptor 2 phosphorylation and c-Jun N-terminal kinase phosphorylation. Moreover, the VEGF-mediated phosphorylations of endothelial nitric oxide synthase, protein kinase B, and extracellular signal-regulated kinase were obviously decreased by (3 ± 0.37)-, (2 ± 0.27)- and (6 ± 0.23)-fold, respectively, in the presence of resveratrol at high concentration. Parallelly, the VEGF-induced reactive oxygen species formation was significantly decreased by 50 ± 7.88% to 120 ± 14.82% under resveratrol treatment. Thus, our results provided support to the antiangiogenic roles of resveratrol, as well as its related signaling mechanisms, in attenuating the VEGF-mediated responses. The present results supported possible development of resveratrol, which should be considered as a therapeutic agent in terms of prevention and clinical treatment of diseases related to angiogenesis.

Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria, Serpentes).

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP-cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.

Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos.

Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine embryos. The present study examined the onset of mitochondrial (mt) DNA replication in equine embryos and investigated whether IVEP affects the timing of this important event, or the expression of genes required for mtDNA replication (i.e. mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and single-stranded DNA binding protein (SSB)). We also investigated whether developmental arrest was associated with low mtDNA copy number. mtDNA copy number increased (P<0.01) between the early and expanded blastocyst stages both invivo and invitro, whereas the mtDNA:total DNA ratio was higher in invitro-produced embryos (P=0.041). Mitochondrial replication was preceded by an increase in TFAM but, unexpectedly, not mtPOLB or SSB expression. There was no association between embryonic arrest and lower mtDNA copy numbers.

The N terminus of Ascl1 underlies differing proneural activity of mouse and Xenopus Ascl1 proteins

The proneural basic-helix-loop-helix (bHLH) transcription factor Ascl1 is a master regulator of neurogenesis in both central and peripheral nervous systems in vivo, and is a central driver of neuronal reprogramming in vitro. Over the last three decades, assaying primary neuron formation in Xenopus embryos in response to transcription factor overexpression has contributed to our understanding of the roles and regulation of proneural proteins like Ascl1, with homologues from different species usually exhibiting similar functional effects. Here we demonstrate that the mouse Ascl1 protein is twice as active as the Xenopus protein in inducing neural-β-tubulin expression in Xenopus embryos, despite there being little difference in protein accumulation or ability to undergo phosphorylation, two properties known to influence Ascl1 function. This superior activity of the mouse compared to the Xenopus protein is dependent on the presence of the non-conserved N terminal region of the protein, and indicates species-specific regulation that may necessitate care when interpreting results in cross-species experiments.