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At least 19 recordsLinked to original sources
PubMed2025

Foxi2 and Sox3 are master regulators controlling ectoderm germ layer specification.

In vertebrates, germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomics analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification.

Ep300↗
PubMed2025

Foxi2 and Sox3 are master transcription regulators that control ectoderm germ layer specification in Xenopus.

Germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomic analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification.

Animals↗
PubMed2025

Foxh1 is a locus-specific PRC2 recruiter governing germ layer silencing.

Polycomb Repressive Complex 2 (PRC2) establishes H3K27me3 marks to shape spatiotemporal gene expression during embryogenesis. While its dysregulation is linked to developmental disorders, cancer, and aging, the mechanisms guiding PRC2 to specific genomic loci remain a subject of ongoing debate. A prevailing model proposes that PRC2 recruitment occurs via its intrinsic affinity for chromatin rather than through sequence-specific transcription factors. Here, we provide evidence that the maternally deposited pioneer transcription factor Foxh1 plays a critical role in directing PRC2 to specific genomic loci during zygotic genome activation in Xenopus. Foxh1 is a critical transcription factor mediating Nodal signaling, but it also plays an earlier role by pre-binding enhancers prior to signaling activation. This pre-binding is essential for forming enhanceosome complexes that trigger mesendodermal gene expression and drive gastrulation, in cooperation with other maternal transcription factors. Using maternal Foxh1-null embryos, we demonstrate that Foxh1 directly recruits Ezh2, the catalytic subunit of PRC2, to Foxh1-bound loci. Loss of Foxh1 impairs Ezh2 recruitment, leading to a global reduction in H3K27me3. These findings support a dual-function model in which Foxh1 not only activates endodermal gene expression in endoderm, but also recruits PRC2 to silence the same genes in ectoderm. This dual activity of Foxh1 allows the spatially coordinated epigenetic states of the endodermal gene regulatory program during early embryogenesis.

CRISPR/Cas9↗
PubMed2025

Enhancer remodeling by OTX2 directs specification and patterning of mammalian definitive endoderm.

The molecular mechanisms that drive essential patterning events in the mammalian embryo remain poorly understood. Analysis of transcription factor expression kinetics at peri-gastrulation stages of development suggest Otx2 as a candidate regulator of the definitive endoderm, the precursor of all gut-derived organs. Accordingly, timed OTX2 depletion in gastruloids or during directed differentiation results in abnormal definitive endoderm specification in mouse and human, characterized by altered expression of components and transcriptional targets of the canonical WNT signaling pathway, perturbed adhesion and migration programs, and de-repression of regulators of other lineages. These defects cumulate in impaired foregut formation. Mechanistically, OTX2 is required to activate a subset of endoderm-specific enhancers and to suppress select enhancers of other lineages, allowing timely exit from the primitive streak and correct specification of anterior endoderm. Our results establish OTX2 as an early gut regulator and suggest molecular principles underlying spatiotemporal cell identity conserved across germ layers and species.

Otx Transcription Factors↗
PubMed1976

Delineation of La Crosse virus in developmental stages of transovarially infected Aedes triseriatus.

The tropisms and development of La Crosse (LAC) virus in stages of transovarially infected Aedes triseriatus were studied with fluorescent antibody (FA) stained dissected organs and titrations of individual arthropods in suckling mice. Viral antigen was detected by FA in 95 of 387 dissected larvae, pupae, and adults. In larvae highest levels of fluorescence were detected in the alimentary tract, followed by ganglia, malpighian tubule, muscle, and other tissues. No specific organs or germ layer-derived tissues appeared to be the sole source of viral replication. Most tissues and organs of A. triseriatus are capable of maintaining LAC virus. Antigen was detected in the identifiable organs immediately upon emergence from the egg. In pupae and adults antigen was detected at high levels in foregut, gonadal and associated tissues, and in salivary glands, which would indicate females could be infective upon emergence. Virus was isolated from all arthropod stages, in 32 of 130 individuals inoculated into suckling mice. Titrations ranged from less than 1.0 log10 SMICLD50 per 0.02 ml for eggs and 1st instar larvae to 3.0 log10 SMICLD50 for 4th instar larvae. Adults and pupae averaged between 2.0 and 3.0 log10 SMICLD50. Increases in titer during maturation were mainly related to increases in size of the organism rather than in titer per unit volume.

Aedes↗
PubMed2025

Glycolytic activity instructs germ layer proportions through regulation of Nodal and Wnt signaling.

Metabolic pathways can influence cell fate decisions, yet their regulative role during embryonic development remains poorly understood. Here, we demonstrate an instructive role of glycolytic activity in regulating signaling pathways involved in mesoderm and endoderm specification. Using a mouse embryonic stem cell (mESC)-based in vitro model for gastrulation, we found that glycolysis inhibition increases ectodermal cell fates at the expense of mesodermal and endodermal lineages. We demonstrate that this relationship is dose dependent, enabling metabolic control of germ layer proportions through exogenous glucose levels. We further show that glycolysis acts as an upstream regulator of Nodal and Wnt signaling and that its influence on cell fate specification can be decoupled from its effects on growth. Finally, we confirm the generality of our findings using a human gastrulation model. Our work underscores the dependence of signaling pathways on metabolic conditions and provides mechanistic insight into the nutritional regulation of cell fate decision-making.

Glycolysis↗
PubMed1978

Inhibition of growth in vitro by glucocorticoids in mouse embryonic facial mesenchyme cells.

The growth of primary embryonic facial mesenchyme cells established from cleft palate sensitive A/J and resistant C57BL/6J (C57) mice is inhibited by glucocorticoid treatment. A reduction in cell number in both A/J and C57 culture is accompanied by a significant decrease in [3H] thymidine incorporation into both acid soluble and insoluble material. No significant changes in total cellular protein or [14C] leucine incorporation were observed in either cell type. A greater reduction in [3H] thymidine incorporation occurs in cells undergoing exponential growth following steroid exposure than in cells approaching stationary growth. In both A/J and C57 cultures the reduction in cell number exhibits a dose-dependent response to dexamethasone; is specific for glucocorticoids; and is dependent upon the concentration of serum in which the cells are maintained. A/J cells show a greater sensitivity to the inhibitory effect of dexamethasone on cell number and thymidine incorporation than comparably treated C57 cells. Specific, high affinity, saturable cytoplasmic receptors for [3H] dexamethasone are present in the maxillary cytosols from which the primary cultures were established. These receptors exhibit binding specificity for glucocorticoids, and have properties which are similar to glucocorticoid receptors identified in other systems. In both cell types, a correlation exists between the degree of growth inhibition or reduction of [3H] thymidine incorporation and the level of glucocorticoid receptors. These results provide evidence for a receptor-mediated set of responses to glucocorticoids in these cells.

Animals↗
PubMed1975

Cystic degeneration of the telencephalic subependymal germinal layer in newborn infants.

Cystic lesions were found in the telencephalic germinal layer of 12 newborn babies. According to their location, the cysts could be divided into three groups: anterior, middle or thalamostriate, and posterior. The histological appearance of all cysts was essentially the same, but in three cases the germinal layer had a peculiar alveolar type of microcystic degeneration. A constant feature was the presence in the cyst wall of small white granulations composed of germinal cells and/or glial tissue. Cystic degeneration of the germinal layer was usually bilateral and sometimes quite extensive. After the involution of the germinal layer, these lesions are likely to persist as subependymal cysts, characterized by their specific location and the presence of glial granulations.

Brain Diseases↗
PubMed1978

A specific dimerization of rabbit beta-globin messenger ribonucleic acid.

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.

Animals↗
PubMed2027

Teratoma Formation and Genomic Profiling Using Multi-Omics Approaches.

Teratoma formation is the gold standard assay for evaluating the developmental pluripotency of human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Following subcutaneous injection into immunodeficient mice, pluripotent stem cells spontaneously differentiate into derivatives representing all three embryonic germ layers-ectoderm, mesoderm, and endoderm. Beyond serving as a functional assay for pluripotency, teratomas provide a unique three-dimensional model system for studying early human development and lineage specification in vivo. This chapter describes comprehensive protocols for teratoma formation in immunodeficient mice, tissue processing for multiple downstream genomic applications, and multi-omics profiling approaches. We detail methods for embryonic stem cell culture, teratoma generation via subcutaneous injection, tissue dissection and processing for chromatin immunoprecipitation followed by sequencing (ChIP-Seq), RNA sequencing (RNA-Seq), single-cell multiome profiling combining chromatin accessibility (ATAC-Seq) and gene expression (scRNA-Seq), and histological analysis using hematoxylin and eosin (H&E) staining. Additionally, we provide bioinformatics workflows for analyzing the resulting genomic datasets to characterize the epigenetic and transcriptional landscapes of teratoma-derived tissues. These methods enable comprehensive molecular characterization of developmental processes and provide valuable resources for stem cell biologists studying pluripotency, differentiation, and early embryonic development.

Teratoma↗
PubMed1979

Ecological determinants in microbial colonization of the murine gastrointestinal tract: adherence of Torulopsis pintolopesii to epithelial surfaces.

Torulopsis pintolopesii is a yeast indigenous to the gastrointestinal tracts of conventional mice and rats from many colonies. In such natively colonized animals, the organism forms layers on the surface of the epithelium in the secreting portion of the stomach and can be cultured from all areas of the gastrointestinal tract. When given in water or food to germfree mice or specific pathogen-free mice possessing an indigenous microbiota free of yeast, T. pintolopesii also can be cultured from all areas of the tract at population levels ranging from 10(5) to 10(8) cells per g (wet weight). Likewise, as in its native hosts, the organism forms layers on gastric surfaces in the associated animals. The layers form on the secreting surface in both the specific pathogen-free and monoassociated ex-germfree mice. In the latter animal, however, a layer of yeast also forms on the nonsecreting gastric surface. In tests of its capacity to adhere to gastrointestinal surfaces in vitro, the organism adheres to epithelia from all areas of the mouse tract. These findings support an hypothesis that the capacity of T. pintolopesii to adhere to epithelial surfaces may be only one determinant influencing it to form layers on the gastric secreting surface in its native hosts.

Animals↗
PubMed1978

Localization of alpha-galactomannan and of wheat germ agglutinin receptors in Schizosaccharomyces pombe.

The location of galactomannan on the surface of Schizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized on S. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections of S. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed that S. pombe cell wall is devoid of chitin.

Ascomycota↗
PubMed1976

In vitro development of secondary blastodiscs from dispersed blastoderm cells of Gallus domesticus.

A study of the in vitro growth of embryonic structures from dispersed blastoderm cells is reported. The specific type of blastoderm cells with the capability of growing on the coverslip developed into the discoidal embryonic structures resembling those of avian species. The glass surface was apparently an initiator of differentiation into at least three types of cells specifically distributed in the blastodisc. Groups of structures formed were evaluated at 6, 12, 24 and 36 hours to study the developmental pattern in vitro and to estimate the number of cells per structure. Microscopic examination of the area pellucida revealed that all three basic germ layers were established after 24 hours of incubation in vitro. The 36 hour stage was represented by bulky growth of mesodermal-like cells and changes in hypoblast layer where some of the cells degenerated and some were transformed to mesenchymal spindle-like cells.

Animals↗
PubMed1978

Investigation of the effect of hydroxyurea on the cell cycle and the development of necrosis in the embryonic CNS of mice.

On day 10 of gestation pregnant mice (strain NMRI) were given an intravenous injection of 500 mg/kg Hydroxyurea (HU). Simultaneously either 5 muCi/g or 10 muCi/g 3H-thymidine (3H-Tdr; specific activity 5Ci/mmol) was administered to the animals. At various times after treatment embryos and electron microscopy. Two hours after administration of HU condensations of the chromatin structure could be detected electron microscopically in some cells. Thirty minutes later the nucleolus became smaller and denser, the cytoplasm shrank, and the cell organelles moved closer together. Three hours after application of the drug break-down of the involved cells set in. As in the autoradiograms about 98% of the counted necroses were labelled, and since labelled thymidine is almost exclusively incorporated during the S-phase, it can be stated the HU influences only metabolic processes which take place during the S-phase. From the morphological findings it can be concluded that in the case of the S-phase-specific metabolic pathway, which is influenced by HU, we are primarily dealing with DNA synthesis.

Animals↗
PubMed1978

[Effect of neuropharmacologic preparations and colchicine on morphogenetic processes in embryonal amphibian cells].

The effect of antagonists of biogenous amines (antitransmitters--AT) and colchicine on rapid morphogenetic processes in the explants of embryonic ectoderm with underlying mesoderm cut from the lateral region of Rana temporaria embryos at the late neurula-early taibud stages was studied. The normal morphogenesis of the explants consists of two successive phases: phase of contact polarization and phase of cell movement into the fragment. The high concentrations of AT inhibited completely the morphogenesis of explants but somewhat lower concentrations inhibited the second phase of morphogenesis and not only did not prevent the cell polarization but even assisted its propagation over all the fragment. The inhibiting effect of AT was relieved by 5-hydroxytryptamine which per se stimulated the morphogenesis of explants. Thus, AT exert a specific inhibition of the motility of embryonic cells but do not prevent the contact interactions responsible for cell polarization within every layer.

Amphetamines↗
PubMed1975

[Action of microbial flora of the digestive tract on the metabolism of bile acids in the rat (author's transl)].

An isotopic balance is established in rats receiving a regular feed intake of [4-14-C]cholesterol so that various chemical species of bile acids have the same specific activity. This property is used to study bile acids distribution in the rat liver, digestive tract and fecal excretion. Bile acids are separated by thin-layer chromatography, radioactivity is determined by liquid scintillation, and the mass by 3-hydroxysteroid dehydrogenase action. The resulting comparative study made between the germ-free rat (axenic rat) and the rat exposed to microbes ("holoxenic" or conventional rat) receiving a semi-synthetetic feed, shows the influence excercised on the metabolism by the microbial flora of the digestive tract. This study confirms that the axenic rat compared to its holoxenic homologue has a higher bile acids pool and a lower fecal excretion. At all levels of the digestive tract (small intestine and the whole caecum and large intestine), probably as well as in the liver, the total amount of bile acids which is observed in the axenic rat is about twice the amount observed in the holoxenic rat, but fecal excretion is decreased by 20%. Values obtained by this method are higher than those previously observed by other authors using gas-liquid chromatography or [14-C]cholic acid isotopic dilution. This study also confirms that cholic and beta-muricholic acids are the main bile acids in the axenic rat and in addition establishes that in this animal bile acids composition is complex and varies from the small intestine to the feces. Besides cholic, alpha- and beta-muricholic, chenodeoxycholic and ursodeoxycholic acids, unidentified chemical species constitute 21% of the whole in the feces. Comparing the compositions observed in axenic and holoxenic rats in this experiment, it could not be determined if the relative activity of the two pathways of bile acid biosynthesis is deeply or only slightly changed by the presence of microbial flora. This is because of a large fraction of unknown composants in the feces of the axenic rat and the extreme complexity in the feces of the holoxenic rat.

Animals↗