Connexin 43 Gene Ablation Does Not Alter Human Pluripotent Stem Cell Germ Lineage Specification

During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell–cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.

The Organizer and Its Signaling in Embryonic Development

Germ layer specification and axis formation are crucial events in embryonic development. The Spemann organizer regulates the early developmental processes by multiple regulatory mechanisms. This review focuses on the responsive signaling in organizer formation and how the organizer orchestrates the germ layer specification in vertebrates. Accumulated evidence indicates that the organizer influences embryonic development by dual signaling. Two parallel processes, the migration of the organizer’s cells, followed by the transcriptional activation/deactivation of target genes, and the diffusion of secreting molecules, collectively direct the early development. Finally, we take an in-depth look at active signaling that originates from the organizer and involves germ layer specification and patterning.

Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula

Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on chrd (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits chrd expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the chrd promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the chrd promoter. Site-directed mutagenesis of FRE within the chrd(-2250) promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates chrd transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of chrd transcription during neuroectoderm and mesoderm germ-layer specification in Xenopus embryos.

Dissemination of echinococcus in the abdominal and pelvic cavity. I. I. Chizhov (Tr. I Congress of Khir. North-Caucasian Territories)

I. I. Chizhov (Tr. I Congress of Khir. North Kavk. Territory) for. to determine about what elements of the opened echinococcal bladder the seeding occurs, put experiments on rabbits and found that all the elements that have fallen out of the echinococcal cyst can be implanted in the abdominal cavity and in the pelvic cavity, and only those formations that carry in themselves can grow and develop supply of the living germ layer of the echinococcal parasite.

Functional Roles of FGF Signaling in Early Development of Vertebrate Embryos

Fibroblast growth factors (FGFs) comprise a large family of growth factors, regulating diverse biological processes including cell proliferation, migration, and differentiation. Each FGF binds to a set of FGF receptors to initiate certain intracellular signaling molecules. Accumulated evidence suggests that in early development and adult state of vertebrates, FGFs also play exclusive and context dependent roles. Although FGFs have been the focus of research for therapeutic approaches in cancer, cardiovascular disease, and metabolic syndrome, in this review, we mainly focused on their role in germ layer specification and axis patterning during early vertebrate embryogenesis. We discussed the functional roles of FGFs and their interacting partners as part of the gene regulatory network for germ layer specification, dorsal–ventral (DV), and anterior-posterior (AP) patterning. Finally, we briefly reviewed the regulatory molecules and pharmacological agents discovered that may allow modulation of FGF signaling in research.

Study of the Growth and Development of Pterocarya pterocarpa (Michx.) Kunth ex Iljinsk. in ex situ Conditions

Results of analyzes of the growth and development of species Pterocarya pterocarpa (Michx.) Kunth ex Iljinsk. of the Pterocarya Kunth genus (Juglandaceae DC. ex Perleb) — relict plant of the Arctic flora of the III period in ex situ conditions have been shown in the paper. Degree of adaptation of wingnut in ex situ conditions, its growth characteristics were assessed both in young and old age. The biomorphological structure of P. pterocarpa seedlings, the size and number of hypocotyls and germ layer were determined in the course of the study. The formation and growth of simple complex true leaves and the life cycle of germ layer were determined as a result of observations of seedlings. The growth of seedlings of wingnut occurs intensively from the third decade of April to the second decade of June under ex situ conditions have been showed the study. The final productivity of seedlings under in situ conditions was higher than under cultivation conditions in ex situ as was found under studying the growth and development of plant. The vegetation of the species P. pterocarpa under the conditions of Absheron begins from the second decade of March to the second decade of November; the duration of the growing season is several days. P. pterocarpa grows and develops normally in cultural conditions of Absheron on based of the analysis of the research.

Optimization of the TeraTox assay for preclinical teratogenicity assessment

Teratogenicity poses severe threats to patient safety. Stem-cell-based in vitro systems are promising tools to predict human teratogenicity. However, current in vitro assays are limited because they either capture effects on a certain germ layer, or focus on a subset of predictive markers. Here we report the characterization and critical assessment of TeraTox, a newly developed multi-lineage differentiation assay using 3D human induced pluripotent stem cells. TeraTox probes stem-cell derived embryoid bodies with two endpoints, one quantifying cytotoxicity and the other inferring the teratogenicity potential with gene expression as a molecular phenotypic readout. To derive teratogenicity potentials from gene expression profiles, we applied both unsupervised machine-learning tools including factor analysis and supervised tools including classification and regression. To identify the best predictive model for the teratogenicity potential that is explainable, we systematically tested 64 machine-learning model architectures and identified the optimal model, which uses expression of 77 representative germ-layer genes, summarized by 10 latent germ-layer factors, as input for random-forest regression. We combined measured cytotoxicity and inferred teratogenicity potential to predict concentration-dependent teratogenicity profiles of 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Compared with the mouse embryonic stem cell test, which has been in routine use for more than a decade, the TeraTox assay shows higher sensitivity, particularly towards teratogens impairing ectodermal development or stem-cell renewal, and a more balanced prediction performance. We envision that further refinement and development of TeraTox has the potential to reduce and replace animal research in drug discovery and to improve preclinical assessment of teratogenicity.

Epithelial cell plasticity drives endoderm formation during gastrulation

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock‐in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.