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.

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.

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.

Charting human development using a multi-endodermal organ atlas and organoid models.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

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.

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.

Mesodermal FGF and BMP govern the sequential stages of zebrafish thyroid specification.

Thyroid tissue, the site of de novo thyroid hormone biosynthesis, is derived from ventral pharyngeal endoderm and defects in morphogenesis are a predominant cause of congenital thyroid diseases. The first molecularly recognizable step of thyroid development is the specification of thyroid precursors in anterior foregut endoderm. Recent studies have identified crucial roles of FGF and BMP signaling in thyroid specification, but the interplay between signaling cues and thyroid transcription factors remained elusive. By analyzing Pax2a and Nkx2.4b expression dynamics in relation to endodermal FGF and BMP signaling activities in zebrafish embryos, we identified a Pax2a-expressing thyroid progenitor population that shows enhanced FGF signaling but lacks Nkx2.4b expression and BMP signaling. Concurrent with upregulated BMP signaling, a subpopulation of these progenitors subsequently differentiates into lineage-committed thyroid precursors co-expressing Pax2a and Nkx2.4b. Timed manipulation of FGF/BMP activities suggests a model in which FGF signaling primarily regulates Pax2a expression, whereas BMP signaling regulates both Pax2a and Nkx2.4b expression. Our observation of similar expression dynamics of Pax8 and Nkx2-1 in mouse embryos suggests that this refined model of thyroid cell specification is evolutionarily conserved in mammals.

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.

TMEM132A regulates mouse hindgut morphogenesis and caudal development.

Caudal developmental defects, including caudal regression, caudal dysgenesis and sirenomelia, are devastating conditions affecting the skeletal, nervous, digestive, reproductive and excretory systems. Defects in mesodermal migration and blood supply to the caudal region have been identified as possible causes of caudal developmental defects, but neither satisfactorily explains the structural malformations in all three germ layers. Here, we describe caudal developmental defects in transmembrane protein 132a (Tmem132a) mutant mice, including skeletal, posterior neural tube closure, genitourinary tract and hindgut defects. We show that, in Tmem132a mutant embryos, visceral endoderm fails to be excluded from the medial region of early hindgut, leading directly to the loss or malformation of cloaca-derived genitourinary and gastrointestinal structures, and indirectly to the neural tube and kidney/ureter defects. We find that TMEM132A mediates intercellular interaction, and physically interacts with planar cell polarity (PCP) regulators CELSR1 and FZD6. Genetically, Tmem132a regulates neural tube closure synergistically with another PCP regulator Vangl2. In summary, we have identified Tmem132a as a new regulator of PCP, and hindgut malformation as the underlying cause of developmental defects in multiple caudal structures.

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.

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells.

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids.

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.

The GATA factor ELT-3 specifies endoderm in Caenorhabditis angaria in an ancestral gene network.

Endoderm specification in Caenorhabditis elegans occurs through a network in which maternally provided SKN-1/Nrf, with additional input from POP-1/TCF, activates the GATA factor cascade MED-1,2→END-1,3→ELT-2,7. Orthologues of the MED, END and ELT-7 factors are found only among nematodes closely related to C. elegans, raising the question of how gut is specified in their absence in more distant species in the genus. We find that the C. angaria, C. portoensis and C. monodelphis orthologues of the GATA factor gene elt-3 are expressed in the early E lineage, just before their elt-2 orthologues. In C. angaria, Can-pop-1(RNAi), Can-elt-3(RNAi) and a Can-elt-3 null mutation result in a penetrant 'gutless' phenotype. Can-pop-1 is necessary for Can-elt-3 activation, showing that it acts upstream. Forced early E lineage expression of Can-elt-3 in C. elegans can direct the expression of a Can-elt-2 transgene and rescue an elt-7 end-1 end-3; elt-2 quadruple mutant strain to viability. Our results demonstrate an ancestral mechanism for gut specification and differentiation in Caenorhabditis involving a simpler POP-1→ELT-3→ELT-2 gene network.

H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate.

Mouse embryonic stem cells have an inherent propensity to explore gene regulatory states associated with either self-renewal or differentiation. This property depends on ERK, which downregulates pluripotency genes such as Nanog. Here, we aimed at identifying repressive histone modifications that would mark Nanog for inactivation in response to ERK activity. We found that the transcription factor ZFP57, which binds methylated DNA to nucleate heterochromatin, is recruited upstream of Nanog, within a region enriched for histone H3 lysine 9 tri-methylation (H3K9me3). Whereas before differentiation H3K9me3 at Nanog depends on ERK, in somatic cells it becomes independent of ERK. Moreover, the loss of H3K9me3 at Nanog, induced by deleting the region or by knocking out DNA methyltransferases or Zfp57, is associated with reduced heterogeneity of NANOG, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. Hence, a network axis centred on DNA methylation, ZFP57 and H3K9me3 links Nanog regulation to ERK activity for the timely establishment of new cell identities. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.

Feedforward regulatory logic controls the specification-to-differentiation transition and terminal cell fate during Caenorhabditis elegans endoderm development.

The architecture of gene regulatory networks determines the specificity and fidelity of developmental outcomes. We report that the core regulatory circuitry for endoderm development in Caenorhabditis elegans operates through a transcriptional cascade consisting of six sequentially expressed GATA-type factors that act in a recursive series of interlocked feedforward modules. This structure results in sequential redundancy, in which removal of a single factor or multiple alternate factors in the cascade leads to a mild or no effect on gut development, whereas elimination of any two sequential factors invariably causes a strong phenotype. The phenotypic strength is successfully predicted with a computational model based on the timing and levels of transcriptional states. We found that one factor in the middle of the cascade, END-1, which straddles the distinct events of specification and differentiation, functions in both processes. Finally, we reveal roles for key GATA factors in establishing spatial regulatory state domains by repressing other fates, thereby defining boundaries in the digestive tract. Our findings provide a paradigm that could account for the genetic redundancy observed in many developmental regulatory systems.

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.

Alliance of Heart and Endoderm: Multilineage Organoids to Model Co-development.

Studies in animal models tracing organogenesis of the mesoderm-derived heart have emphasized the importance of signals coming from adjacent endodermal tissues in coordinating proper cardiac morphogenesis. Although in vitro models such as cardiac organoids have shown great potential to recapitulate the physiology of the human heart, they are unable to capture the complex crosstalk that takes place between the co-developing heart and endodermal organs, partly due to their distinct germ layer origins. In an effort to address this long-sought challenge, recent reports of multilineage organoids comprising both cardiac and endodermal derivatives have energized the efforts to understand how inter-organ, cross-lineage communications influence their respective morphogenesis. These co-differentiation systems have produced intriguing findings of shared signaling requirements for inducing cardiac specification together with primitive foregut, pulmonary, or intestinal lineages. Overall, these multilineage cardiac organoids offer an unprecedented window into human development that can reveal how the endoderm and heart cooperate to direct morphogenesis, patterning, and maturation. Further, through spatiotemporal reorganization, the co-emerged multilineage cells self-assemble into distinct compartments as seen in the cardiac-foregut, cardiac-intestine, and cardiopulmonary organoids and undergo cell migration and tissue reorganization to establish tissue boundaries. Looking into the future, these cardiac incorporated, multilineage organoids will inspire future strategies for improved cell sourcing for regenerative interventions and provide more effective models for disease investigation and drug testing. In this review, we will introduce the developmental context of coordinated heart and endoderm morphogenesis, discuss strategies for in vitro co-induction of cardiac and endodermal derivatives, and finally comment on the challenges and exciting new research directions enabled by this breakthrough.

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.