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.

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.

Cell surface fluctuations regulate early embryonic lineage sorting.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells.

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.

A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming.

Somatic cells can be reprogrammed into pluripotent stem cells (PSCs) by using pure chemicals, providing a different paradigm to study somatic reprogramming. However, the cell fate dynamics and molecular events that occur during the chemical reprogramming process remain unclear. We now show that the chemical reprogramming process requires the early formation of extra-embryonic endoderm (XEN)-like cells and a late transition from XEN-like cells to chemically-induced (Ci)PSCs, a unique route that fundamentally differs from the pathway of transcription factor-induced reprogramming. Moreover, precise manipulation of the cell fate transition in a step-wise manner through the XEN-like state allows us to identify small-molecule boosters and establish a robust chemical reprogramming system with a yield up to 1,000-fold greater than that of the previously reported protocol. These findings demonstrate that chemical reprogramming is a promising approach to manipulate cell fates.

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.

Interaction domains of Sos1/Grb2 are finely tuned for cooperative control of embryonic stem cell fate.

Metazoan evolution involves increasing protein domain complexity, but how this relates to control of biological decisions remains uncertain. The Ras guanine nucleotide exchange factor (RasGEF) Sos1 and its adaptor Grb2 are multidomain proteins that couple fibroblast growth factor (FGF) signaling to activation of the Ras-Erk pathway during mammalian development and drive embryonic stem cells toward the primitive endoderm (PrE) lineage. We show that the ability of Sos1/Grb2 to appropriately regulate pluripotency and differentiation factors and to initiate PrE development requires collective binding of multiple Sos1/Grb2 domains to their protein and phospholipid ligands. This provides a cooperative system that only allows lineage commitment when all ligand-binding domains are occupied. Furthermore, our results indicate that the interaction domains of Sos1 and Grb2 have evolved so as to bind ligands not with maximal strength but with specificities and affinities that maintain cooperativity. This optimized system ensures that PrE lineage commitment occurs in a timely and selective manner during embryogenesis.

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.

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.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

Gata6 functions in zebrafish endoderm to regulate late differentiating arterial pole cardiogenesis.

Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24 h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation.

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.

Gα13 controls pharyngeal endoderm convergence by regulating E-cadherin expression and RhoA activation.

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.

CellRank 2: unified fate mapping in multiview single-cell data.

Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies.

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.

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.

HBO1 determines SMAD action in pluripotency and mesendoderm specification.

TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.

A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo.

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.

Enhancer, transcriptional, and cell fate plasticity precedes intestinal determination during endoderm development.

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure.

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.