Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5-8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4-labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

The body cavity is a space where internal organs develop and are placed. Despite the importance of this internal space, how the body cavity forms specifically within the mesoderm remains largely unknown. Here, we report that upon the onset of dorsal mesodermal cell polarization and initial lumen formation, mesodermal cells form filamentous projections that are directed toward the ectoderm. This cell behavior enables the dorsal population of mesodermal cells to receive BMP7 that is expressed by the ectoderm. Suppression of ectodermal BMP7 diminishes mesodermal cell projection and results in the loss of body cavity development. The data reveal that body cavity induction depends on signaling factor transfer from ectoderm to mesoderm.

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Preclinical and clinical evidence implicates N-methyl-d-aspartate receptor (NMDAr) signaling in early embryological development. However, the role of NMDAr signaling in early development has not been well studied. Here, we use a mouse embryonic stem cell model to perform a step-wise exploration of the effects of NMDAr signaling on early cell fate specification. We found that antagonism of the NMDAr impaired specification into the neuroectodermal and mesoendodermal cell lineages, with little or no effect on specification of the extraembryonic endoderm cell lineage. Consistent with these findings, exogenous NMDA promoted neuroectodermal differentiation. Finally, NMDAr antagonism modified expression of several key targets of TGF-β superfamily signaling, suggesting a mechanism for these findings. In summary, this study shows that NMDAr antagonism interferes with the normal developmental pathways of embryogenesis, and suggests that interference is most pronounced prior to neuroectodermal and mesoendodermal cell fate specification.

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions.

Li-Fraumeni syndrome is caused by germline TP53 mutations and is clinically characterized by a predisposition to a range of cancers, most commonly sarcoma, brain tumours and leukemia. Pathogenic mosaic TP53 mutations have only rarely been described.

DNA methylation of regulatory and growth-related genes contributes to fetal programming which is important for maintaining the correct development of three germ layers of the embryo that develope into different tissues and organs, and which persists into adult life. In this study, a preliminary epigenetic screen was performed to define genomic regions that are involved in fetal epigenome remodeling. Embryonic ectodermic tissues (origin of nervous tissue), mesenchymal tissues (origin of connective and muscular tissues), and foregut endoderm tissues (origin of epithelial tissue), from day 28 sheep fetuses were collected and the distribution of methylated CpGs was analyzed using whole-genome bisulfite sequencing. Patterns of methylation among the three tissues showed a high level of conservation of hypo-methylated CpG islands CGIs, and a consistent level of methylation in regulatory genetic elements. Analysis of tissue specific differentially methylated regions, revealed that 20% of the total CGIs differed between tissues. A proportion of the methylome was remodeled in gene bodies, 5' UTRs and 3' UTRs (7, 11, and 11%, respectively). Genes with overlapping differentially methylated regions in gene bodies and CGIs showed a significant enrichment for tissue morphogenesis and development pathways. The data presented here provides a "reference" for the epigenetic status of genes potentially involved in the maintenance and regulation of fetal developmental during early life, a period expected to be particularly prone to epigenetic alterations induced by environmental and nutritional stressors.

Heterochronic blood sharing rejuvenates old tissues, and most of the studies on how this works focus on young plasma, its fractions, and a few youthful systemic candidates. However, it was not formally established that young blood is necessary for this multi-tissue rejuvenation. Here, using our recently developed small animal blood exchange process, we replaced half of the plasma in mice with saline containing 5% albumin (terming it a "neutral" age blood exchange, NBE) thus diluting the plasma factors and replenishing the albumin that would be diminished if only saline was used. Our data demonstrate that a single NBE suffices to meet or exceed the rejuvenative effects of enhancing muscle repair, reducing liver adiposity and fibrosis, and increasing hippocampal neurogenesis in old mice, all the key outcomes seen after blood heterochronicity. Comparative proteomic analysis on serum from NBE, and from a similar human clinical procedure of therapeutic plasma exchange (TPE), revealed a molecular re-setting of the systemic signaling milieu, interestingly, elevating the levels of some proteins, which broadly coordinate tissue maintenance and repair and promote immune responses. Moreover, a single TPE yielded functional blood rejuvenation, abrogating the typical old serum inhibition of progenitor cell proliferation. Ectopically added albumin does not seem to be the sole determinant of such rejuvenation, and levels of albumin do not decrease with age nor are increased by NBE/TPE. A model of action (supported by a large body of published data) is that significant dilution of autoregulatory proteins that crosstalk to multiple signaling pathways (with their own feedback loops) would, through changes in gene expression, have long-lasting molecular and functional effects that are consistent with our observations. This work improves our understanding of the systemic paradigms of multi-tissue rejuvenation and suggest a novel and immediate use of the FDA approved TPE for improving the health and resilience of older people.

In animal embryos, gene regulatory networks control the dynamics of gene expression in cells and coordinate such dynamics among cells. In ascidian embryos, gene expression dynamics have been dissected at the single-cell resolution. Here, we revealed mathematical functions that represent the regulatory logics of all regulatory genes expressed at the 32-cell stage when the germ layers are largely specified. These functions collectively explain the entire mechanism by which gene expression dynamics are controlled coordinately in early embryos. We found that regulatory functions for genes expressed in each of the specific lineages contain a common core regulatory mechanism. Last, we showed that the expression of the regulatory genes became reproducible by calculation and controllable by experimental manipulations. Thus, these regulatory functions represent an architectural design for the germ layer specification of this chordate and provide a platform for simulations and experiments to understand the operating principles of gene regulatory networks.

Much progress has been made toward generating analogs of early embryos, such as gastruloids and embryoids, in vitro. However, methods for how to fully mimic the cell movements of gastrulation and coordinate germ-layer patterning to induce head formation are still lacking. Here, we show that a regional Nodal gradient applied to zebrafish animal pole explant can generate a structure that recapitulates the key cell movements of gastrulation. Using single-cell transcriptome and in situ hybridization analysis, we assess the dynamics of the cell fates and patterning of this structure. The mesendoderm differentiates into the anterior endoderm, prechordal plate, notochord, and tailbud-like cells along an anterior-posterior axis, and an anterior-posterior-patterned head-like structure (HLS) progressively forms during late gastrulation. Among 105 immediate Nodal targets, 14 genes contain axis-induction ability, and 5 of them induce a complete or partial head structure when overexpressed in the ventral side of zebrafish embryos.

Specification of germ layers along the dorsoventral axis by morphogenetic gradients is an ideal model to study scaling properties of gradients and cell fate changes during evolution. Classical anatomical studies in divergent insects (e.g., flies and grasshoppers) revealed that the neuroectodermal size is conserved and originates similar numbers of neuroblasts of homologous identity. In contrast, mesodermal domains vary significantly in closely related Drosophila species. To further investigate the underlying mechanisms of scaling of germ layers across Drosophila species, we quantified the Dorsal (Dl)/NF-κB gradient, the main morphogenetic gradient that initiates separation of the mesoderm, neuroectoderm, and ectoderm. We discovered a variable range of Toll activation across species and found that Dl activates mesodermal genes at the same threshold levels in melanogaster sibling species. We also show that the Dl gradient distribution can be modulated by nuclear size and packing densities. We propose that variation in mesodermal size occurs at a fast evolutionary rate and is an important mechanism to define the ventral boundary of the neuroectoderm.

Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4-/-) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4-/- cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4-/- iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells.

Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells.

Human organoid model systems lack important cell types that, in the embryo, are incorporated into organ tissues during development. We developed an organoid assembly approach starting with cells from the three primary germ layers-enteric neuroglial, mesenchymal, and epithelial precursors-that were derived separately from human pluripotent stem cells (PSCs). From these three cell types, we generated human antral and fundic gastric tissue containing differentiated glands surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered antral tissue. Using this experimental system, we show that human enteric neural crest cells (ENCCs) promote mesenchyme development and glandular morphogenesis of antral stomach organoids. Moreover, ENCCs can act directly on the foregut to promote a posterior fate, resulting in organoids with a Brunner's gland phenotype. Thus, germ layer components that are derived separately from PSCs can be used for tissue engineering to generate complex human organoids.

In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.