A geometrical model of cell fate specification in the mouse blastocyst.

The lineage decision that generates the epiblast and primitive endoderm from the inner cell mass (ICM) is a paradigm for cell fate specification. Recent mathematics has formalized Waddington's landscape metaphor and proven that lineage decisions in detailed gene network models must conform to a small list of low-dimensional stereotypic changes called bifurcations. The most plausible bifurcation for the ICM is the so-called heteroclinic flip that we define and elaborate here. Our re-analysis of recent data suggests that there is sufficient cell movement in the ICM so the FGF signal, which drives the lineage decision, can be treated as spatially uniform. We thus extend the bifurcation model for a single cell to the entire ICM by means of a self-consistently defined time-dependent FGF signal. This model is consistent with available data and we propose additional dynamic experiments to test it further. This demonstrates that simplified, quantitative and intuitively transparent descriptions are possible when attention is shifted from specific genes to lineages. The flip bifurcation is a very plausible model for any situation where the embryo needs control over the relative proportions of two fates by a morphogen feedback.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation.

BACKGROUND: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. METHODS: The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. RESULTS: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. CONCLUSIONS: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect.

Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sublethal dose of pathogens or nonpathogenic microbes stimulates immunity and prevents subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, monospecies Caballeronia symbiont population in the posterior region. We demonstrate that a particular Burkholderia strain colonizing the anterior midgut stimulates systemic immunity by penetrating gut epithelia and migrating into the hemolymph. The activated immunity, consisting of a humoral and a cellular response, had no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia strain and epithelia of the gut weakened the host immunity back to preinfection levels and made the insects more vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity.

Gastrulation: Its Principles and Variations.

As epiblast cells initiate development into various somatic cells, they undergo a large-scale reorganization, called gastrulation. The gastrulation of the epiblast cells produces three groups of cells: the endoderm layer, the collection of miscellaneous mesodermal tissues, and the ectodermal layer, which includes the neural, epidermal, and associated tissues. Most studies of gastrulation have focused on the formation of the tissues that provide the primary route for cell reorganization, that is, the primitive streak, in the chicken and mouse. In contrast, how gastrulation alters epiblast-derived cells has remained underinvestigated. This chapter highlights the regulation of cell and tissue fate via the gastrulation process. The roles and regulatory functions of neuromesodermal progenitors (NMPs) in the gastrulation process, elucidated in the last decade, are discussed in depth to resolve points of confusion. Chicken and mouse embryos, which form a primitive streak as the site of mesoderm precursor ingression, have been investigated extensively. However, primitive streak formation is an exception, even among amniotes. The roles of gastrulation processes in generating various somatic tissues will be discussed broadly.

Glycolysis-Stimulated Esrrb Lactylation Promotes the Self-Renewal and Extraembryonic Endoderm Stem Cell Differentiation of Embryonic Stem Cells.

Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance, and exit. However, an understanding of how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. The lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of the LIF and XEN differentiation of ESCs by increasing its binding at target genes. Our studies reveal the importance of protein lactylation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism of glycolytic metabolism regulating cell fate choice.

SOX17 enables immune evasion of early colorectal adenomas and cancers.

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1-3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5- tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids.

Human primordial germ cells (hPGCs), the precursors of eggs and sperm, start their complex development shortly after specification and during their migration to the primitive gonads. Here, we describe protocols for specifying hPGC-like cells (hPGCLCs) from resetting precursors and progressing them with the support of human hindgut organoids. Resetting hPGCLCs (rhPGCLCs) are specified from human embryonic stem cells (hESCs) transitioning from the primed into the naive state of pluripotency. Hindgut organoids are also derived from hESCs after a sequential differentiation into a posterior endoderm/hindgut fate. Both rhPGCLCs and hindgut organoids are combined and co-cultured for 25 d. The entire procedure takes ~1.5 months and can be successfully implemented by a doctoral or graduate student with basic skills and experience in hESC cultures. The co-culture system supports the progression of rhPGCLCs at a developmental timing analogous to that observed in vivo. Compared with previously developed hPGCLC progression protocols, which depend on co-cultures with mouse embryonic gonadal tissue, our co-culture system represents a developmentally relevant model closer to the environment that hPGCs first encounter after specification. Together with the potential for investigations of events during hPGC specification and early development, these protocols provide a practical approach to designing efficient models for in vitro gametogenesis. Notably, the rhPGCLC-hindgut co-culture system can also be adapted to study failings in hPGC migration, which are associated with the etiology of some forms of infertility and germ cell tumors.

Molecular profiling of human blastocysts reveals primitive endoderm defects among embryos of decreased implantation potential.

Human embryo implantation is remarkably inefficient, and implantation failure remains among the greatest obstacles in treating infertility. Gene expression data from human embryos have accumulated rapidly in recent years; however, identification of the subset of genes that determine successful implantation remains a challenge. We leverage clinical morphologic grading-known for decades to correlate with implantation potential-and transcriptome analyses of matched embryonic and abembryonic samples to identify factors and pathways enriched and depleted in human blastocysts of good and poor morphology. Unexpectedly, we discovered that the greatest difference was in the state of extraembryonic primitive endoderm (PrE) development, with relative deficiencies in poor morphology blastocysts. Our results suggest that implantation success is most strongly influenced by the embryonic compartment and that deficient PrE development is common among embryos with decreased implantation potential. Our study provides a valuable resource for those investigating the markers and mechanisms of human embryo implantation.

Transcription factor co-expression mediates lineage priming for embryonic and extra-embryonic differentiation.

In early mammalian development, cleavage stage blastomeres and inner cell mass (ICM) cells co-express embryonic and extra-embryonic transcriptional determinants. Using a protein-based double reporter we identify an embryonic stem cell (ESC) population that co-expresses the extra-embryonic factor GATA6 alongside the embryonic factor SOX2. Based on single cell transcriptomics, we find this population resembles the unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence. To relate transcription factor binding in these cells to future fate, we describe a complete enhancer set in both ESCs and naive extra-embryonic endoderm stem cells and assess SOX2 and GATA6 binding at these elements in the ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers, suggesting that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.

Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

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.

PAR-4/LKB1 prevents intestinal hyperplasia by restricting endoderm specification in Caenorhabditis elegans embryos.

The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation.

Thyroid Hormone Levels Correlate With the Maturation of Implanted Pancreatic Endoderm Cells in Patients With Type 1 Diabetes.

BACKGROUND: Macroencapsulated pancreatic endoderm cells (PECs) can reverse diabetes in rodents and preclinical studies revealed that thyroid hormones in vitro and in vivo bias PECs to differentiate into insulin-producing cells. In an ongoing clinical trial, PECs implanted in macroencapsulation devices into patients with type 1 diabetes were safe but yielded heterogeneous outcomes. Though most patients developed meal responsive C-peptide, levels were heterogeneous and explanted grafts had variable numbers of surviving cells with variable distribution of endocrine cells. METHODS: We measured circulating triiodothyronine and thyroxine levels in all patients treated at 1 of the 7 sites of the ongoing clinical trial and determined if thyroid hormone levels were associated with the C-peptide or glucagon levels and cell fate of implanted PECs. RESULTS: Both triiodothyronine and thyroxine levels were significantly associated with the proportion of cells that adopted an insulin-producing fate with a mature phenotype. Thyroid hormone levels were inversely correlated to circulating glucagon levels after implantation, suggesting that thyroid hormones lead PECs to favor an insulin-producing fate over a glucagon-producing fate. In mice, hyperthyroidism led to more rapid maturation of PECs into insulin-producing cells similar in phenotype to PECs in euthyroid mice. CONCLUSION: These data highlight the relevance of thyroid hormones in the context of PEC therapy in patients with type 1 diabetes and suggest that a thyroid hormone adjuvant therapy may optimize cell outcomes in some PEC recipients.

Fgf signalling is required for gill slit formation in the skate, Leucoraja erinacea.

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm-ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm-ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal-ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.

Automated, High-Throughput Phenotypic Screening and Analysis Platform to Study Pre- and Post-Implantation Morphogenesis in Stem Cell-Derived Embryo-Like Structures.

Combining high-throughput generation and high-content imaging of embryo models will enable large-scale screening assays in the fields of (embryo) toxicity, drug development, embryogenesis, and reproductive medicine. This study shows the continuous culture and in situ (i.e., in microwell) imaging-based readout of a 3D stem cell-based model of peri-implantation epiblast (Epi)/extraembryonic endoderm (XEn) development with an expanded pro-amniotic cavity (PAC) (E3.5 E5.5), namely XEn/EPiCs. Automated image analysis and supervised machine learning permit the identification of embryonic morphogenesis, tissue compartmentalization, cell differentiation, and consecutive classification. Screens with signaling pathway modulators at different time windows provide spatiotemporal information on their phenotypic effect on developmental processes leading to the formation of XEn/EPiCs. Exposure of the biological model in the microwell platform to pathway modulators at two time windows, namely 0-72 h and 48-120 h, show that Wnt and Fgf/MAPK pathway modulators affect Epi differentiation and its polarization, while modulation of BMP and Tgfß/Nodal pathway affects XEn specification and epithelialization. Further, their collective role is identified in the timing of the formation and expansion of PAC. The newly developed, scalable culture and analysis platform, thereby, provides a unique opportunity to quantitatively and systematically study effects of pathway modulators on early embryonic development.

Uncovering Phenotypes in Mutant Mice by Determining Embryo, Organ, Tissue, and Cell Developmental Potential.

The death of an embryo during gestation does not necessarily preclude the study of the mutant embryo or the developmental potential of its individual cells, tissues, or organs. Whole-embryo in vitro culture prior to the time of death will allow real-time observation of living embryos and direct comparisons with controls. Organ anlage can be removed from embryos and cultured in vitro beyond the time of death of the whole embryo. In both whole embryos and organ anlage culture, fluorescent protein reporters may be used productively to follow cell types or specific gene expression changes. Some cells, such as hematopoietic cells, and organ anlage, may be suitable for transplantation to wild-type hosts for further analysis of their potential. Additionally, cell lines, including embryonic stem (ES) cells, trophoblast stem (TS) cells, extraembryonic endoderm (XEN) stem cells, and epiblast-derived stem cells (EpiSC), can be derived from mutant embryos to reveal the potential of the mutant cells outside the context of the whole organism. Mutant stem cells or even whole mutant embryos can be used to test potential in chimeras or in teratomas.

Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture.

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

A Comparative Study of Endoderm Differentiation Between Activin A and Small Molecules.

Small molecules such as ROCK inhibitors (Fasudil) and inducer of definitive endoderm 1 (IDE1) can promote differentiation of definitive endoderm, but their effects remain controversial. Therefore, we attempted to verify the effect of these small molecules on promoting definitive endoderm differentiation and found that Fasudil or IDE1 alone could not achieve a similar effect as activin A. On the contrary, CHIR99021 could efficiently promote definitive endoderm differentiation. Nearly 43.4% of experimental cells were SRY-box transcription factor 17 (SOX17)-positive under the synergistic effect of IDE1 and CHIR99021, but its ability to differentiate towards definitive endoderm was still insufficient. Transcriptional analysis and comparison of IDE1 and CHIR99021 synergistic groups (IC) and activin A and CHIR99021 synergistic groups (AC) showed significantly down-regulated definitive endoderm markers in the IC group compared with those in the AC group and the differences between the two groups were mainly due to bone morphogenetic proteins (BMP4) and fibroblast growth factor 17 (FGF17). Further single-cell transcriptome analysis revealed lower expression of BMP4 in SOX17-positive populations, while mothers against decapentaplegic homolog (SMAD) protein translation signal and FGF17 in the AC group were higher than that in the IC group. Western blot analysis showed a significant difference in levels of p-SMAD2/3 between AC and IC groups, which suggests that regulating p-SMAD2/3 may provide a reference to improve the differentiation of definitive endoderm.

A chemo-mechanical model of endoderm movements driving elongation of the amniote hindgut.

Although mechanical and biochemical descriptions of development are each essential, integration of upstream morphogenic cues with downstream tissue mechanics remains understudied during vertebrate morphogenesis. Here, we developed a two-dimensional chemo-mechanical model to investigate how mechanical properties of the endoderm and transport properties of fibroblast growth factor (FGF) regulate avian hindgut morphogenesis in a coordinated manner. Posterior endoderm cells convert a gradient of FGF ligands into a contractile force gradient, leading to a force imbalance that drives collective cell movements that elongate the forming hindgut tube. We formulated a 2D reaction-diffusion-advection model describing the formation of an FGF protein gradient as a result of posterior displacement of cells transcribing unstable Fgf8 mRNA during axis elongation, coupled with translation, diffusion and degradation of FGF protein. The endoderm was modeled as an active viscous fluid that generates contractile stresses in proportion to FGF concentration. With parameter values constrained by experimental data, the model replicates key aspects of hindgut morphogenesis, suggests that graded isotropic contraction is sufficient to generate large anisotropic cell movements, and provides new insight into how chemo-mechanical coupling across the mesoderm and endoderm coordinates hindgut elongation with axis elongation.