Transcriptional and epigenetic characterization of a new in vitro platform to model the formation of human pharyngeal endoderm.

BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.

A pre-vertebrate endodermal origin of calcitonin-producing neuroendocrine cells.

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.

A Three-Step Protocol to Differentiate iPSC into Colon Organoids.

Organoids, three-dimensional, stem cell-based structures that mimic the cellular and functional architecture of tissues, have emerged as an innovative in vitro tool. They offer highly efficient models for studying both embryonic development and disease progression processes. Colon organoids can also be generated from biopsies obtained during a colonoscopy. However, the invasive nature of biopsy collection poses practical challenges and introduces biases when studying patients who are already afflicted. Therefore, the use of iPSC-derived colon organoids can be considered a more practical approach for researchers and patients alike. Numerous protocols have been published for generating colon organoids from iPSCs. While most of these protocols share a common developmental process, some are labor-intensive or require additional equipment. Taking these considerations into account, we present a cost-effective and straightforward yet functionally robust colon organoid protocol: (1) definitive endoderm differentiation, (2) hindgut endoderm differentiation, and (3) maturation of colon spheroids into mature organoids.

Cell size regulates human endoderm specification through actomyosin-dependent AMOT-YAP signaling.

Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.

Toward developing human organs via embryo models and chimeras.

Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.

An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration.

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Differential retinoic acid sensitivity of oral and pharyngeal teeth in medaka (Oryzias latipes) supports the importance of pouch-cleft contacts in pharyngeal tooth initiation.

BACKGROUND: Previous studies have claimed that pharyngeal teeth in medaka (Oryzias latipes) are induced independent of retinoic acid (RA) signaling, unlike in zebrafish (Danio rerio). In zebrafish, pharyngeal tooth formation depends on a proper physical contact between the embryonic endodermal pouch anterior to the site of tooth formation, and the adjacent ectodermal cleft, an RA-dependent process. Here, we test the hypothesis that a proper pouch-cleft contact is required for pharyngeal tooth formation in embryonic medaka, as it is in zebrafish. We used 4-[diethylamino]benzaldehyde (DEAB) to pharmacologically inhibit RA production, and thus pouch-cleft contacts, in experiments strictly controlled in time, and analyzed these using high-resolution imaging. RESULTS: Pharyngeal teeth in medaka were present only when the corresponding anterior pouch had reached the ectoderm (i.e., a physical pouch-cleft contact established), similar to the situation in zebrafish. Oral teeth were present even when the treatment started approximately 4 days before normal oral tooth appearance. CONCLUSIONS: RA dependency for pharyngeal tooth formation is not different between zebrafish and medaka. We propose that the differential response to DEAB of oral versus pharyngeal teeth in medaka could be ascribed to the distinct germ layer origin of the epithelia involved in tooth formation in these two regions.

Coordination between endoderm progression and mouse gastruloid elongation controls endodermal morphotype choice.

Embryonic development is highly robust. Morphogenetic variability between embryos (under ideal conditions) is largely quantitative. This robustness stands in contrast to in vitro embryo-like models, which, like most organoids, can display a high degree of tissue morphogenetic variability. The source of this difference is not fully understood. We use the mouse gastruloid model to study the morphogenetic progression of definitive endoderm (DE) and its divergence. We first catalog the different morphologies and characterize their statistics. We then learn predictive models for DE morphotype based on earlier expression and morphology measurements. Finally, we analyze these models to identify key drivers of morphotype variability and devise gastruloid-specific and global interventions that can lower this variability and steer morphotype choice. In the process, we identify two types of coordination lacking in the in vitro model but required for robust gut-tube formation. This approach can help improve the quality and usability of 3D embryo-like models.

Neural Crest.

This chapter discusses the role of cardiac neural crest cells in the formation of the septum that divides the cardiac arterial pole into separate systemic and pulmonary arteries. Further, cardiac neural crest cells directly support the normal development and patterning of derivatives of the caudal pharyngeal arches, including the great arteries, thymus, thyroid, and parathyroids. Recently, cardiac neural crest cells have also been shown to indirectly influence the development of the secondary heart field, another derivative of the caudal pharynx, by modulating signaling in the pharynx. The contribution and function of the cardiac neural crest cells has been learned in avian models; most of the genes associated with cardiac neural crest function have been identified using mouse models. Together these studies show that the neural crest cells may not only critical for normal cardiovascular development but also may be involved secondarily because they represent a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Cardiac neural crest cells span from the caudal pharynx into the outflow tract, and therefore may be susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations resulting from genetic and/or environmental insults necessarily requires better understanding the role of cardiac neural crest cells in cardiac development.

Sturgeon gut development: a unique yolk utilization strategy among vertebrates.

In vertebrates, maternally supplied yolk is typically used in one of two ways: either intracellularly by endodermal cells or extracellularly via the yolk sac. This study delves into the distinctive gut development in sturgeons, which are among the most ancient extant fish groups, contrasting it with that of other vertebrates. Our observations indicate that while sturgeon endodermal cells form the archenteron (i.e., the primitive gut) dorsally, the floor of the archenteron is uniquely composed of extraembryonic yolk cells (YCs). As development progresses, during neurulation, the archenteric cavity inflates, expands laterally, and roofs a semicircle of YCs. By the pharyngula stage, the cavity fully encompasses the YC mass, which begins to be digested at the hatching stage. This suggests a notable deviation in sturgeon gut development from that in other vertebrates, as their digestive tract initiates its function by processing endogenous nutrition even before external feeding begins. Our findings highlight the evolutionary diversity of gut development strategies among vertebrates and provide new insights into the developmental biology of sturgeons.

The primitive endoderm supports lineage plasticity to enable regulative development.

Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.

Low concentrations of saracatinib promote definitive endoderm differentiation through inhibition of FAK-YAP signaling axis.

Optimizing the efficiency of definitive endoderm (DE) differentiation is necessary for the generation of diverse organ-like structures. In this study, we used the small molecule inhibitor saracatinib (SAR) to enhance DE differentiation of human embryonic stem cells and induced pluripotent stem cells. SAR significantly improved DE differentiation efficiency at low concentrations. The interaction between SAR and Focal Adhesion Kinase (FAK) was explored through RNA-seq and molecular docking simulations, which further supported the inhibition of DE differentiation by p-FAK overexpression in SAR-treated cells. In addition, we found that SAR inhibited the nuclear translocation of Yes-associated protein (YAP), a downstream effector of FAK, which promoted DE differentiation. Moreover, the addition of SAR enabled a significant reduction in activin A (AA) from 50 to 10 ng/mL without compromising DE differentiation efficiency. For induction of the pancreatic lineage, 10 ng/ml AA combined with SAR at the DE differentiation stage yielded a comparative number of PDX1+/NKX6.1+ pancreatic progenitor cells to those obtained by 50 ng/ml AA treatment. Our study highlights SAR as a potential modulator that facilitates the cost-effective generation of DE cells and provides insight into the orchestration of cell fate determination.

RFX6 regulates human intestinal patterning and function upstream of PDX1.

The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.

The migration pattern of cells during the mesoderm and endoderm differentiation from human pluripotent stem cells.

Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell-derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement and external force, such as the spread out of cells from the primitive streak area, mimicked the homogeneous discoidal germ layer formation. These results were consistent with the development of amniotes, which suggests the effectiveness of human pluripotent stem cells as a good model for studying human embryogenesis.

Application of tissue-scale tension to avian epithelia in vivo to study multiscale mechanical properties and inter-germ layer coupling.

As cross-disciplinary approaches drawing from physics and mechanics have increasingly influenced our understanding of morphogenesis, the tools available to measure and perturb physical aspects of embryonic development have expanded as well. However, it remains a challenge to measure mechanical properties and apply exogenous tissue-scale forces in vivo, particularly for epithelia. Exploiting the size and accessibility of the developing chick embryo, here we describe a simple technique to quantitatively apply exogenous forces on the order of ~1-100 µN to the endodermal epithelium. To demonstrate the utility of this approach, we performed a series of proof-of-concept experiments that reveal fundamental and unexpected mechanical behaviors in the early chick embryo, including mechanotype heterogeneity among cells of the midgut endoderm, complex non-cell autonomous effects of actin disruption, and a high degree of mechanical coupling between the endoderm and adjacent paraxial mesoderm. To illustrate the broader utility of this method, we determined that forces on the order of ~ 10 µN are sufficient to unzip the neural tube during primary neurulation. Together, these findings provide basic insights into the mechanics of embryonic epithelia in vivo in the early avian embryo, and provide a useful tool for future investigations of how morphogenesis is influenced by mechanical factors.

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.

Tissues and signals with true organizer properties in craniofacial development.

Transplantation experiments have shown that a true organizer provides instructive signals that induce and pattern ectopic structures in the responding tissue. Here, we review craniofacial experiments to identify tissues with organizer properties and signals with organizer properties. In particular, we evaluate whether transformation of identity took place in the mesenchyme. Using these stringent criteria, we find the strongest evidence for the avian foregut ectoderm. Transplanting a piece of quail foregut endoderm to a host chicken embryo caused ectopic beaks to form derived from chicken mesenchyme. The beak identity, whether upper or lower as well as orientation, was controlled by the original anterior-posterior position of the donor endoderm. There is also good evidence that the nasal pit is necessary and sufficient for lateral nasal patterning. Finally, we review signals that have organizer properties on their own without the need for tissue transplants. Mouse germline knockouts of the endothelin pathway result in transformation of identity of the mandible into a maxilla. Application of noggin-soaked beads to post-migratory neural crest cells transforms maxillary identity. This suggests that endothelin or noggin rich ectoderm could be organizers (not tested). In conclusion, craniofacial, neural crest-derived mesenchyme is competent to respond to tissues with organizer properties, also originating in the head. In future, we can exploit such well defined systems to dissect the molecular changes that ultimately lead to patterning of the upper and lower jaw.

Assessment of human embryonic stem cells differentiation into definitive endoderm lineage on the soft substrates.

Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells (hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein (YAP) and phosphorylated YAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.

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.

OCT4 is expressed in extraembryonic endoderm stem (XEN) cell progenitors during somatic cell reprogramming.

During development, progenitors of embryonic stem (ES) and extraembryonic endoderm stem (XEN) cells are concomitantly specified within the inner cell mass (ICM) of the mouse blastocyst. Similarly, XEN cells are induced (iXEN cells) alongside induced pluripotent stem (iPS) cells following overexpression of Oct4, Sox2, Klf4 and Myc (OSKM) during somatic cell reprogramming. It is unclear how or why this cocktail produces both stem cell types, but OCT4 has been associated with non-pluripotent outcomes. In this report, we show that, during OSKM reprogramming, many individual Oct4-GFP-expressing cells are fated to become iXEN cells. Interestingly, SKM alone was also sufficient to induce iXEN cell formation, likely via activation of endogenous Oct4. These observations indicate that iXEN cell formation is not strictly an artifact of Oct4 overexpression. Moreover, our results suggest that a pathway to XEN may be an integral feature of establishing pluripotency during reprogramming, as in early embryo development.