Role of Alpha-Fetoprotein in the Pathogenesis of Cancer.

Alpha-fetoprotein (AFP) belongs to the albuminoid protein family and is considered as the fetal analog of serum albumin. This plasma protein is initially synthesized in the fetal liver and yolk sac and shows a maximum peak near the end of the first trimester. Later, concentrations begin to decline prenatally and drop precipitously after birth. This protein has three key ligand-binding pockets for interactions with various biomolecules. It contains multiple phosphorylation and acetylation sites for the regulation of physiological and pathophysiological states. High serum AFP titer is an established biomarker for yolk sac, embryonal and hepatocellular carcinoma. The present review critically analyzes the chemical nature, receptors, clinical implications, and therapeutic aspects of AFP, underpinning the development of different types of cancer.

Metabolomics analysis of the yolk of Zhijin white goose during the embryogenesis based on LC-MS/MS.

The egg yolk of the goose is rich in lipids, proteins and minerals, which is the main source of nutrition during the goose embryogenesis. Actually, the magnitude and variety of nutrients in yolk are dynamically changed to satisfy the nutritional requirements of different growth and development periods. The yolk sac membrane (YSM) plays a role in metabolizing and absorbing nutrients from the yolk, which are then consumed by the embryo or extra-fetal tissues. Therefore, identification of metabolites in egg yolk can help to reveal nutrient requirement in goose embryo. In this research, to explore the metabolite changes in egg yolk at embryonic day (E) 7, E12, E18, E23, and E28, we performed the assay using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The findings showed that E7 and E12, E23 and E28 were grouped together, while E18 was significantly separated from other groups, indicating the changes of egg yolk development and metabolism. In total, 1472 metabolites were identified in the egg yolk of Zhijin white goose, and 636 differential metabolites (DMs) were screened, among which 264 were upregulated and 372 were downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the DMs were enriched in the biosynthesis and metabolism of amino acids, digestion and absorption of protein, citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, phosphotransferase system (PTS), mineral absorption, cholesterol metabolism and pyrimidine metabolism. Our study may provide new ideas for improving prehatch embryonic health and nutrition.

In ovo injection of testosterone to yolk sac modulates early posthatching development and physiology of male chick in broilers.

The aim of this study was to investigate the effects of in ovo testosterone injection into the yolk sac of embryos on physiology and development of broiler chicks during the early posthatching period. A total of 1,010 hatching eggs were obtained from the Ross genotype. Trial design was conducted with a noninjected group (control) and injection groups in which 100 µL sesame oil, or 100 µL sesame oil + 0.50 µmol testosterone were injected into the yolk sac of the embryo on d 6 or d 12 of incubation. Testosterone hormone level was measured in the egg yolk and albumen at onset of incubation, in the yolk sac on d 19 of incubation and in the residual yolk sac at hatching. Weights of chick, yolk sac and organ, morphological traits (body length, lengths of bilateral traits and beak length), asymmetrical development of bilateral morphological traits and body mass index were measured at hatching and on d 7 after hatching. Testosterone, corticosterone and growth hormone levels were determined in blood plasma obtained from male chicks at hatching and on d 7 of chick age. Chick weight was not affected, plasma testosterone level and brain weight decreased, while body mass index, plasma corticosterone and growth hormone levels increased by administering 0.50 µmol testosterone on d 12 of embryonic age. However, plasma testosterone and growth hormone levels did not change, chick weight increased, while plasma corticosterone level and the chick body length decreased by administering 0.50 µmol testosterone on d 6 of embryonic age. A significant interaction between chick age and in ovo testosterone administration resulted in an increase in lung weight of chicks. In conclusion, this study found that in ovo testosterone administered at different embryonic ages due to age-specific effects of testosterone in the yolk sac of embryo modulates development related to physiological parameters of male broiler chicks during early posthatching period.

Developmental Associations between Neurovascularization and Microglia Colonization.

The temporal and spatial pattern of microglia colonization and vascular infiltration of the nervous system implies critical associated roles in early stages of nervous system development. Adding to existing reviews that cover a broad spectrum of the various roles of microglia during brain development, the current review will focus on the developmental ontogeny and interdependency between the colonization of the nervous system with yolk sac derived macrophages and vascularization. Gaining a better understanding of the timing and the interdependency of these two processes will significantly contribute to the interpretation of data generated regarding alterations in either process during early development. Additionally, such knowledge should provide a framework for understanding the influence of the early gestational environmental and the impact of genetics, disease, disorders, or exposures on the early developing nervous system and the potential for long-term and life-time effects.

Effects of water fluoridation on early embryonic development of zebrafish.

Fluoride has strong electronegativity and exposes diversely in nature. Water fluoridation is the most pervasive form of occurrence, representing a significant threat to human health. In this study, we investigate the morphometric and physiological alterations triggered by fluoride stimulation during the embryogenesis of zebrafish and reveal its putative effects of stage- and/or dose-dependent. Fluoride exhibits potent biological activity and can be extensively absorbed by the yolk sac, exerting significant effects on the development of multiple organs. This is primarily manifested as restricted nutrient utilization and elevated levels of lipid peroxidation, further leading to the accumulation of superoxide in the yolk sac, liver, and intestines. Moreover, pericardial edema exerts pressure on the brain and eye development, resulting in spinal curvature and reduced body length. Besides, acute fluoride exposure with varying concentrations has led to diverse teratogenic outcomes. A low dose of water fluoridation tends to induce abnormal development of the embryonic yolk sac, while vascular malformation is widely observed in all fluoride-treated groups. The effect of fluoride exposure on blood circulation is universally present, even in zebrafish larvae that do not exhibit obvious deformities. Their swimming behavior is also affected by water fluoridation, resulting in reduced activity and delayed reactions. In conclusion, this study provides valuable insights into the monitoring of environmental quality related to water fluoridation and disease prevention.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

Fate-Mapping of Yolk Sac-Derived Macrophages.

To better understand the distinct functions of yolk-sac-derived tissue-resident macrophages (TRMs) and bone-marrow-derived macrophages in homeostasis and disease, it is important to trace the ontogeny of these cells. The majority of TRMs originate from erythro-myeloid progenitors (EMPs). EMPs develop into pre-macrophages (pMacs), which can be detected starting at embryonic developmental day (E)9.0, and which give rise to all TRM during early development. pMacs start expressing the gene Cx3cr1, allowing us to genetically target the early yolk-sac wave of pMacs and their progeny. Here, we describe the protocol for the identification of yolk sac-derived TRMs utilizing in utero labelling of the inducible fate mapping Cx3cr1CreERT; Rosa26LSL-eYFP mouse model.

Apoptotic mechanism of development inhibition in zebrafish induced by esketamine.

Esketamine, a widely used intravenous general anesthetic, is also employed for obstetric and pediatric anesthesia, and depression treatment. However, concerns regarding esketamine abuse have emerged. Moreover, the potential in vivo toxicity of esketamine on growth and development remains unclear. To address these concerns, we investigated the effects of esketamine exposure on developmental parameters, cell apoptosis, and gene expression in zebrafish. Esketamine exposure concentration-dependently decreased the heart rate and body length of zebrafish embryos/larvae while increasing the hatching rate and spontaneous movement frequency. Developmental retardation of zebrafish larvae, including shallow pigmentation, small eyes, and delayed yolk sac absorption, was also observed following esketamine treatment. Esketamine exposure altered the expression of apoptosis-related genes in zebrafish heads, primarily downregulating bax, caspase9, caspase3, caspase6, and caspase7. Intriguingly, BTSA1, a Bax agonist, reversed the anti-apoptotic and decelerated body growth effects of esketamine in zebrafish. Collectively, our findings suggest that esketamine may hinder embryonic development by inhibiting embryonic apoptosis via the Bax/Caspase9/Caspase3 pathway. To the best of our knowledge, this is the first study to report the lethal toxicity of esketamine in zebrafish. We have elucidated the developmental toxic effects of esketamine on zebrafish larvae and its potential apoptotic mechanisms. Further studies are warranted to evaluate the safety of esketamine in animals and humans.

Single-cell RNA sequencing-guided fate-mapping toolkit delineates the contribution of yolk sac erythro-myeloid progenitors.

Erythro-myeloid progenitors of the yolk sac that originates during early embryo development has been suggested to generate tissue-resident macrophage, mast cell, and even endothelial cell populations from fetal to adult stages. However, the heterogeneity of erythro-myeloid progenitors (EMPs) is not well characterized. Here, we adapt single-cell RNA sequencing to dissect the heterogeneity of EMPs and establish several fate-mapping tools for each EMP subset to trace the contributions of different EMP subsets. We identify two primitive and one definitive EMP subsets from the yolk sac. In addition, we find that primitive EMPs are decoupled from definitive EMPs. Furthermore, we confirm that primitive and definitive EMPs give rise to microglia and other tissue-resident macrophages, respectively. In contrast, only Kit+ Csf1r- primitive EMPs generate endothelial cells transiently during early embryo development. Overall, our results delineate the contribution of yolk sac EMPs more clearly based on the single-cell RNA sequencing (scRNA-seq)-guided fate-mapping toolkit.

Maternal physical activity affects yolk sac size and growth in early pregnancy, but girls and boys use different strategies.

This longitudinal study investigated the impact of actigraphy-measured maternal physical activity on yolk sac size during early development. The yolk sac, a transient extraembryonic organ, plays a crucial role in embryonic development and is involved in metabolism, nutrition, growth, and hematopoiesis. Prospectively collected data from 190 healthy women indicated that their total daily physical activity, including both light and moderate-vigorous activity, was associated with yolk sac growth dynamics depending on embryonic sex and gestational age. Higher preconception maternal physical activity was linked to a larger yolk sac at 7 weeks (95% CI [0.02-0.13 mm]) and a smaller yolk sac at 10 weeks' gestation (95% CI [- 0.18 to - 0.00]) in male embryos; in female embryos, the yolk sac size was increased at 10 weeks' gestation (95% CI [0.06-0.26]) and was, on average, 24% larger than that in male embryos (95% CI [0.12-0.38]). Considering the pattern of other maternal effects on yolk sac size-e.g., body composition and sleep duration-we suggest that physiological yolk sac adaptations occur in short, sex-specific time windows and can be influenced by various maternal factors.

Placental development in the early stages of red-rumped agouti pregnancy (Dasyprocta leporina Linnaeus, 1758).

BACKGROUND: Hystricomorpha rodents display a similar placentation model to humans. The present study was carried out considering the scarcity of information concerning the placental development in agouti. OBJECTIVE: Describe the microscopy of the placenta, subplacenta and yolk sac of agoutis in early pregnancy and report on the inversion of the yolk sac. METHODS: Fifteen females between the 14th-32nd day of gestation were used following euthanasia. Gestational buttons were collected, fixed, processed, stained to optical microscopy or immunohistochemistry. RESULTS: Chorioallantoic placenta (CP) ranged from conical to a half-sphere, as follows: from the 14th to 17th day, the CP displays an inverted "V" shape, predominantly formed by cytotrophoblasts; from 20 to 22 days, formed almost entirely by cytotrophoblasts; at 28 days, a half sphere, with distinct lobes and interlobular area, numerous maternal gaps delimited by syncytiotrophoblasts and trophoblast giant cells; at 32 days, globose and undergoing the maturation process. Subplacenta, located between decidua and CP, initially presents septa consisting of simple columnar epithelium and after 17 days, comprising stratified epithelium. Visceral yolk sac (VYS) is attached to two CP projections between 14 and 17 days, formed by a simple cubic epithelium and inverted. Between 20 and 22 days, the epithelium displays apical villous projections with cytoplasmic vacuoles and a vascularized mesoderm. After the 24th day, the VYS near the placenta is pleated, very vascularized and villous, with decreased villi sizes further away from the placenta. CONCLUSION: The agouti CP displays similar characteristics to other hystricomorpha, including placenta lobulation, a subplacenta and an inverted vitelline placenta.

The autotaxin-LPA axis promotes membrane trafficking and secretion in yolk sac visceral endoderm cells.

Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2 mRNA was abundantly expressed in yolk sac visceral endoderm (VE) cells and that Enpp2-/- mice were lethal at embryonic day 9.5 owing to angiogenic defects in the yolk sac. Enpp2-/- mice showed lysosome fragmentation in VE cells and embryonic abnormalities including allantois malformation, neural tube defects, no axial turning, and head cavity formation. However, whether the defects in endocytic vesicle formation affect membrane trafficking in VE cells remained to be directly examined. In this study, we found that pinocytosis, transcytosis, and secretion of angiogenic factors such as vascular endothelial growth factor and transforming growth factor ß1 were impaired in Enpp2-/- VE cells. Moreover, pharmacologic inhibition of membrane trafficking phenocopied the defects of Enpp2-/- mice. These findings demonstrate that Enpp2 promotes endocytosis and secretion of angiogenic factors in VE cells, thereby regulating angiogenesis/vasculogenesis and embryonic development.

Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

Ultrasonographic features of the regressing yolk sac of normal captive Brown kiwi chicks (Apteryx mantelli) vary with age.

Brown kiwi (Apteryx mantelli) are a culturally significant ratite species endemic to New Zealand. Chicks can develop diseases of the yolk sac which can be fatal if not promptly recognized and treated. Ultrasound examination of the yolk sac is an important component in the evaluation of chicks with suspected yolk sac disease. However, there are currently no reports of the normal ultrasonographic appearance of the regressing yolk sac in this species. The objective of this prospective reference interval study was to describe the normal ultrasonographic appearance of the yolk sac in brown kiwi chicks. Focused coelomic sonographic examinations were performed on 29 healthy chicks from >1 day of age until the yolk sac was completely absorbed or was <1 mL in volume. 41.6% (57/137) of yolks were round/ovoid in shape and 58.6% (80/137) had lobulated margins with invaginations. All yolks (137/137) were hyperechoic relative to adjacent coelomic structures. The heterogeneity of the echogenicity of the yolk was significantly associated with age (increased heterogeneity in younger birds) and was homogeneous, mildly heterogeneous, and moderately heterogeneous in 33% (46/138), 46% (63/138), and 21% (29/138) of all examinations respectively. Intrasac anechoic pockets were seen in 14% (20/139) of yolk sacs. The rate of yolk sac resorption slowed with age. In all examinations identifying a completely absorbed yolk sac or a yolk sac <1 mL in volume, chicks were less than 21 days old. The results of this study provide a reference of normal for the sonographic appearance of the yolk sac in captive brown kiwi chicks.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Effects of high incubation temperature on tight junction proteins in the yolk sac and small intestine of embryonic broilers.

During the transition from incubation to hatch, the chicks shift from obtaining nutrients from the yolk sac to the intestine. The yolk sac tissue (YST) and small intestine serve as biological barriers between the yolk or gut contents and the blood circulation. These barriers must maintain structural integrity for optimal nutrient uptake as well as protection from pathogens. The objective of this study was to investigate the effect of high incubation temperature on mRNA abundance of the tight junction (TJ) proteins zona occludens 1 (ZO1), occludin (OCLN), claudin 1 (CLDN1), and junctional adhesion molecules A and 2 (JAMA, JAM2) and the heat shock proteins (HSP70 and HSP90) in the YST and small intestine of embryonic broilers. Broiler eggs were incubated at 37.5°C. On embryonic day 12 (E12), half of the eggs were switched to 39.5°C. YST samples were collected from E7 to day of hatch (DOH), while small intestinal samples were collected from E17 to DOH. The temporal expression of TJ protein mRNA from E7 to DOH at 37.5°C and the effect of incubation temperature from E13 to DOH were analyzed by one-way and two-way ANOVA, respectively and Tukey's test. Significance was set at P < 0.05. The temporal expression pattern of ZO1, OCLN, and CLDN1 mRNA showed a pattern of decreased expression from E7 to E13 followed by an increase to DOH. High incubation temperature caused an upregulation of ZO1 and JAM2 mRNA in the YST and small intestine. Using in situ hybridization, OCLN and JAMA mRNA were detected in the epithelial cells of the YST. In addition, JAMA mRNA was detected in epithelial cells of the small intestine, whereas JAM2 mRNA was detected in the vascular system of the villi and lamina propria. In conclusion, the YST expressed mRNA for TJ proteins and high incubation temperature increased ZO1 and JAM2 mRNA. This suggests that the TJ in the vasculature of the YST and intestine is affected by high incubation temperature.

Self-patterning of human stem cells into post-implantation lineages.

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

3D culture of mesenchymal stem cells from the yolk sac to generate intestinal organoid.

Organoids are in vitro models that originated from the three-dimensional culture of stem cells with the ability to reproduce part of the in vivo structural and functional specificities of body organs. Intestinal organoids have great relevance in cell therapy, as they provide more accurate information about tissue composition and architecture in relation to two-dimensional culture, in addition to serving as a study model for host interaction and drug testing. The yolk sac (YS) is a promising source of mesenchymal stem cells (MSCs), which are multipotent stem cells with self-renewal ability and potential to differentiated into mesenchymal lineages. Besides this, the YS is responsible for the formation of intestinal epithelium during embryonic development. Thus, the aim of this study was to verify if the three-dimensional in vitro culture of stem cells derived from the canine YS is capable of developing intestinal organoids. MSCs from the canine YS and gut cells were isolated and characterized, then three-dimensionally cultured in Matrigel. In both cells lineages, spherical organoids were observed and after 10 days the gut cells formed crypt-like buds and villus-like structures. Despite having the same induction of differentiation process and having the expression of intestinal markers, the MSC from the YS morphology was not in the form of crypt budding. The hypothesis is that these cells could generate structures equivalent to the intestinal organoids of the colon that other studies showed formed only spherical structures. The culture of MSC from the YS, as well as the establishment of protocols for 3D cultivation of this tissue, is relevant, as it will serve as a tool in various applications in basic and scientific biology.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells.

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.