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.

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 The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.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.

Yolk sac development, function and role in rodent pregnancy.

During the early phases of embryonic development, the yolk sac serves as an initial placenta in many animal species. While in some, this role subsides around the end of active organogenesis, it continues to have important functions in rodents, alongside the chorio-allantoic placenta. The yolk sac is the initial site of hematopoiesis in many animal species including primates. Cells of epiblastic origin form blood islands that are the forerunners of hematopoietic cells and of the primitive endothelial cells that form the vitelline circulation. The yolk sac is also a major route of embryonic and fetal nutrition apparently as long as it functions. In mammals and especially rodents, macro and micronutrients are absorbed by active pinocytosis into the visceral yolk sac, degraded and the degradation products (i.e., amino acids) are then transferred to the embryo. Interference with the yolk sac function may directly reflect on embryonic growth and development, inducing congenital malformations or in extreme damage, causing embryonic and fetal death. In rodents, many agents were found to damage the yolk sac (i.e., anti-yolk sac antibodies or toxic substances interfering with yolk sac pinocytosis) subsequently affecting the embryo/fetus. Often, the damage to the yolk sac is transient while embryonic damage persists. In humans, decreased yolk sac diameter was associated with diabetic pregnancies and increased diameter was associated with pregnancy loss. In addition, culture of rat yolk sacs in serum obtained from pregnant diabetic women or from women with autoimmune diseases induced severe damage to the visceral yolk sac epithelium and embryonic malformations. It can be concluded that as a result of the crucial role of the yolk sac in the well-being of the early embryo, any damage to its normal function may severely and irreversibly affect further development of the embryo/fetus.

Pure post-pubertal yolk sac tumor of the testis: An extremely rare and aggressive entity.

CONTEXT: Pure post-pubertal yolk sac tumors (YSTs) are an extremely rare type of malignant germ cell tumor (GCT) that account for <1 % of testicular GCTs. Clinically, they are more aggressive compared to the more common pre-pubertal counterpart. The aim of this study is to analyze the clinical presentation, ancillary tests and clinical outcomes in addition to presenting a spectrum of histomorphological features, in a case series along with a literature review. DESIGN: A retrospective review of 4 cases of pure post-pubertal YST of the testis was performed. Data collected for each patient included demographics, clinical presentation, serum markers, radiology and pathologic findings, treatment, and clinical outcomes. RESULTS: All patients presented with a testicular mass with or without associated pain and elevated serum alpha-feto protein. Mean age at presentation was 36 years (range 25-68 years). Two patients presented with metastatic disease at the time of diagnosis. Histologic patterns and features are as follows: germ cell neoplasia in-situ (n = 4), reticular/microcystic, solid, glandular, papillary, endometrioid, cystic, necrosis and angiolymphatic invasion (n = 3). Fluorescent in-situ hybridization test performed on Case 2, showed presence of isochromosome 12p and next generation sequencing showed gains of 12p. Case 1, 2 and 4 showed metastatic disease on follow-up. CONCLUSIONS: Diagnosis of pure post-pubertal YST remains challenging due to the variety of morphologic patterns often present in these tumors. Extensive sampling along with use of ancillary tests is the key for diagnosis. In this study, 75 % of cases had metastatic disease at or after the diagnosis confirming the aggressive nature of this rare entity.

Temporal expression of avian ß defensin 10 and cathelicidins in the yolk sac tissue of broiler and layer embryos.

The yolk sac is a multifunctional organ, which not only participates in nutrient absorption, but also plays an important role in immune function. The objective of this study was to compare the mRNA abundance of avian ß-defensin 10 (AvBD10) and 3 cathelicidins (CATH1, CATH2, and CATH3) in the yolk sac tissue (YST) of commercial broilers and white egg and brown egg commercial layers. AvBD10 and CATH mRNA abundance was analyzed using two-way ANOVA and Tukey's test, with P < 0.05 being considered significant. AvBD10 and CATH mRNA showed similar temporal expression patterns in the YST of both broiler and layers, with an increase from embryonic day (E) 7 to E9 through E13 followed by a decrease to day of hatch. AvBD10 mRNA showed a breed × age interaction with greater expression in the YST of both layers compared to broilers at E9 and E11. CATH1 mRNA was greater in the YST of brown egg layers than broilers. CATH2 mRNA showed a breed × age interaction, with greater expression in the YST of brown egg layers than broilers at E11. CATH3 mRNA showed no difference in the YST between layers and broilers. Because broilers and brown egg layers are genetically related, these results show that selection for production parameters (broiler vs. layer) and not genetic relatedness (white egg layer vs. brown egg layer and broilers) is the basis for the differences in AvBD10, CATH1, and CATH2 mRNA in the YST of broilers and layers. The yolk-free body weights of broiler embryos were greater than that of both brown and white egg layers from E9 to 17. One possible explanation is that the reduced expression of AvBD10, CATH1 and CATH2 mRNA in the YST of broilers compared to layers at E9 and 11 may be due to faster embryonic growth at the expense of host defense peptide expression in broilers compared to layers.

The embryonic zebrafish brain is seeded by a lymphatic-dependent population of mrc1+ microglia precursors.

Microglia are the resident macrophages of the CNS that serve critical roles in brain construction. Although human brains contain microglia by 4 weeks gestation, an understanding of the earliest microglia that seed the brain during its development remains unresolved. Using time-lapse imaging in zebrafish, we discovered a mrc1a+ microglia precursor population that seeds the brain before traditionally described microglia. These early microglia precursors are dependent on lymphatic vasculature that surrounds the brain and are independent of pu1+ yolk sac-derived microglia. Single-cell RNA-sequencing datasets reveal Mrc1+ microglia in the embryonic brains of mice and humans. We then show in zebrafish that these early mrc1a+ microglia precursors preferentially expand during pathophysiological states in development. Taken together, our results identify a critical role of lymphatics in the microglia precursors that seed the early embryonic brain.

Molecular mechanism of nutrient uptake in developing embryos of oviparous cloudy catshark (Scyliorhinus torazame).

Forms of embryonic nutrition are highly diverse in cartilaginous fishes, which contain oviparity, yolk-sac viviparity and several types of matrotrophic viviparity (histotrophy, oophagy, and placentotrophy). The molecular mechanisms of embryonic nutrition are poorly understood in these animals as few species are capable of reproducing in captivity. Oviparous cartilaginous fishes solely depend on yolk nutrients for their growth and development. In the present study, we compared the contribution to embryonic nutrition of the embryonic intestine with the yolk sac membrane (YSM). RNA-seq analysis was performed on the embryonic intestine and YSM of the oviparous cloudy catshark Scyliorhinus torazame to identify candidate genes involved in nutrient metabolism to further the understanding of nutrient utilization of developing embryos. RNA-seq discovery was subsequently confirmed by quantitative PCR analysis and we identified increases in several amino acid transporter genes (slc3a1, slc6a19, slc3a2, slc7a7) as well as genes involved in lipid absorption (apob and mtp) in the intestine after 'pre-hatching', which is a developmental event marked by an early opening of the egg case about 4 months before hatching. Although a reciprocal decrease in the nutritional role of YSM was expected after the intestine became functional, we observed similar increases in gene expression among amino acid transporters, lipid absorption molecules, and lysosomal cathepsins in the extraembryonic YSM in late developmental stages. Ultrastructure of the endodermal cells of YSM showed that yolk granules were incorporated by endocytosis, and the number of granules increased during development. Furthermore, the digestion of yolk granules in the YSM and nutrient transport through the basolateral membrane of the endodermal cells appeared to be enhanced after pre-hatching. These findings suggest that nutrient digestion and absorption is highly activated in both intestine and YSM after pre-hatching in catshark embryos, which supports the rapid growth at late developmental stages.

FOXO1 represses sprouty 2 and sprouty 4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

Lyl-1 regulates primitive macrophages and microglia development.

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/"early EMP" and transient-definitive/"late EMP" waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis.

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors.

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta-gonad-mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

The road (not) taken - Placental transfer and interspecies differences.

Species differences are among the main reasons for the high failure rate of preclinical studies. A better awareness and understanding of these differences might help to improve the outcome of preclinical research. In reproduction, the placenta is the central organ regulating fetal exposure to a substance circulating in the maternal organism. Exact information about placental transfer can help to better estimate the toxic potential of a substance. From an evolutionary point of view, the chorioallantoic placenta is the organ with the highest anatomical diversity among species. Moreover, frequently used animal models in reproduction belong to rodents and lagomorphs, two groups that are characterized by the generation of an additional type of placenta, which is crucial for fetal development, but absent from humans: the inverted yolk sac placenta. Taken together, the translatability of placental transfer studies from laboratory animals to humans is challenging, which is supported by the fact that numerous species-dependent toxic effects are described in literature. Thus, reliable human-relevant data are frequently lacking and the toxic potential of chemicals and pharmaceuticals for humans can hardly be estimated, often resulting in recommendations that medical treatments or exposure to chemicals should be avoided for safety reasons. Although species differences of placental anatomy have been described frequently and the need for human-relevant research models has been emphasized, analyses of substances with species-dependent placental transfer have been performed only sporadically. Here, we present examples for species-specific placental transfer, including that of nanoparticles and pharmaceuticals, and discuss potential underlying mechanisms. With respect to the COVID 19-pandemic it might be of interest that some antiviral drugs are reported to feature species-specific placental transfer. Further, differences in placental structure and antibody transfer may affect placental transfer of ZIKA virus.

VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis.

Exogenous growth factors play an important role in mediating hematopoietic differentiation of human pluripotent stem cells. We explored the role of different factors in early human blood cell production using blast colony formation in methylcellulose as a surrogate assay for yolk sac hematopoiesis. A reporter cell line that read out endothelial (SOX17+) and hematopoietic (RUNX1C+) progenitors facilitated the identification of basic fibroblast growth and vascular endothelial growth factor as critical signals for the progression of mesoderm into endothelium. Bone morphogenetic protein 4 was needed for the subsequent generation of blood from hemogenic endothelium, and this was antagonized by Activin A or high concentrations of the WNT agonist CHIR-99021. Manipulations of the Hedgehog pathway or inhibition of Notch signaling reduced blast colony frequency but did not perturb cell differentiation. These data help to define distinct roles for prerequisite growth factors that commit mesoderm to hemogenic endothelium and subsequently allocate cells to blood lineages.

The effect of an isoquinoline alkaloid on treatment of periodontitis by regulating the neutrophils chemotaxis.

Neutrophil plays a critical role in the progression of periodontitis. In general, its chemotaxis and activation are benefit for the host defense of bacterial infection and inflammation. However, previous studies have reported that the hyperactive and reactive neutrophils appear to be one of the reasons for tissue destruction in periodontitis tissues. In this study, we investigated an isoquinoline alkaloid Litcubanine A (LA), which from the Traditional Chinese medicinal plant, Litsea cubeba. We found LA showed significant activity in inhibiting neutrophils chemotaxis in the zebrafish yolk sac microinjection model in vivo and in mouse neutrophils in vitro. Further investigation proved that LA could inhibit the expression levels of neutrophil respiratory burst-related and inflammation-related genes CYBB and NCF2, as well as inhibit the activation of MAPK signaling pathway. Moreover, using LA, we successfully achieved the effect of reducing periodontitis bone loss by regulating neutrophil chemotaxis and related functions in a mouse ligature-induced periodontitis model.

Spatiotemporal imaging and pharmacokinetics of fluorescent compounds in zebrafish eleuthero-embryos after different routes of administration.

Zebrafish (Danio rerio) is increasingly used to assess the pharmacological activity and toxicity of compounds. The spatiotemporal distribution of seven fluorescent alkyne compounds was examined during 48 h after immersion (10 µM) or microinjection (2 mg/kg) in the pericardial cavity (PC), intraperitoneally (IP) and yolk sac (IY) of 3 dpf zebrafish eleuthero-embryos. By modelling the fluorescence of whole-body contours present in fluorescence images, the main pharmacokinetic (PK) parameter values of the compounds were determined. It was demonstrated that especially in case of short incubations (1-3 h) immersion can result in limited intrabody exposure to compounds. In this case, PC and IP microinjections represent excellent alternatives. Significantly, IY microinjections did not result in a suitable intrabody distribution of the compounds. Performing a QSPkR (quantitative structure-pharmacokinetic relationship) analysis, LogD was identified as the only molecular descriptor that explains the final uptake of the selected compounds. It was also shown that combined administration of compounds (immersion and microinjection) provides a more stable intrabody exposure, at least in case of a prolonged immersion and compounds with LogD value > 1. These results will help reduce the risk of false negative results and can offer an invaluable input for future translational research and safety assessment applications.

The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac.

The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.

Atypical protein kinase C is essential for embryonic vascular development in mice.

The atypical PKC (aPKC) subfamily constitutes PKCζ and PKCλ in mice, and both aPKC isoforms have been proposed to be involved in regulating various endothelial cell (EC) functions. However, the physiological function of aPKC in ECs during embryonic development has not been well understood. To address this question, we utilized Tie2-Cre to delete PKCλ alone (PKCλ-SKO) or both PKCλ and PKCζ (DKO) in ECs, and found that all DKO mice died at around the embryonic day 11.5 (E11.5), whereas a small proportion of PKCλ-SKO mice survived till birth. PKCλ-SKO embryos also exhibited less phenotypic severity than DKO embryos at E10.5 and E11.5, suggesting a potential compensatory role of PKCζ for PKCλ in embryonic ECs. We then focused on DKO embryos and investigated the effects of aPKC deficiency on embryonic vascular development. At E9.5, deletion of both aPKC isoforms reduced the diameters of vitelline artery and vein, and decreased branching from both vitelline vessels in yolk sac. Ablation of both aPKC isoforms also disrupted embryonic angiogenesis in head and trunk at the same stage, increasing apoptosis of both ECs and non-ECs. Taken together, our results demonstrated that aPKC in ECs plays an essential role in regulating cell apoptosis, angiogenesis, and embryonic survival.

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

Epidermal resident γδ T cell development and function in skin.

Epidermal resident γδ T cells, or dendritic epidermal T cells (DETCs) in mice, are a unique and conserved population of γδ T cells enriched in the epidermis, where they serve as the regulators of immune responses and sense skin injury. Despite the great advances in the understanding of the development, homeostasis, and function of DETCs in the past decades, the origin and the underlying molecular mechanisms remain elusive. Here, we reviewed the recent research progress on DETCs, including their origin and homeostasis in the skin, especially at transcriptional and epigenetic levels, and discuss the involvement of DETCs in skin diseases.

Yolk sac-derived Pdcd11-positive cells modulate zebrafish microglia differentiation through the NF-κB-Tgfß1 pathway.

Microglia are the primary immune cells in the central nervous system, which plays a vital role in neuron development and neurodegenerative diseases. Microglial precursors in peripheral hematopoietic tissues colonize the central nervous system during early embryogenesis. However, how intrinsic and extrinsic signals integrate to regulate microglia's differentiation remains undefined. In this study, we identified the cerebral white matter hyperintensities susceptibility gene, programmed cell death protein 11 (PDCD11), as an essential factor regulating microglia differentiation. In zebrafish, pdcd11 deficiency prevents the differentiation of the precursors to mature brain microglia. Although, the inflammatory featured macrophage brain colonization is augmented. At 22 h post fertilization, the Pdcd11-positive cells on the yolk sac are distinct from macrophages and neutrophils. Mechanistically, PDCD11 exerts its physiological role by differentially regulating the functions of nuclear factor-kappa B family members, P65 and c-Rel, suppressing P65-mediated expression of inflammatory cytokines, such as tnfα, and enhancing the c-Rel-dependent appearance of tgfß1. The present study provides novel insights in understanding microglia differentiation during zebrafish development.