Iron restricts radial cadmium transport via Casparian strip development to reduce root-to-shoot translocation in low cadmium-accumulating rice (Oryza sativa L.).

Abstract The transition between a spherical-shaped blastocyst to an elongated conceptus is required for proper early embryonic development in cattle. During the peri-elongation period, embryonic tissues have increased uptake of lipids that will support trophectoderm and endoderm formation. Therefore, our objective was to provide a dietary supplementation strategy containing rumen-inert mono- and polyunsaturated fatty acids and rumen-protected choline to cater to the specific needs of the elongating conceptus. One hundred suckled multiparous Angus cows were randomly assigned on d -30 to receive either TARG) 100 g of a rumen-inert mono- and polyunsaturated fatty acid source (Essentiom; Church and Dwight Co., Inc., Princeton, NJ) plus 60 g of a rumen-protected choline source (ReaShure; Balchem, Montvale, NJ) or CON) 114 g of a saturated fatty acid source (Energy Booster 100; Milk Specialties, Eden Prairie, MN). Treatments were top-dressed daily into a similar total mixed ration until d 30. All cows were synchronized using a 7-day CO-synch+CIDR protocol and received timed artificial insemination by the same technician on d 0. On d 16, uterine flushing was conducted in a subset of cows (CON = 20 and TARG = 23) to determine the presence and length of the embryo, and uterine luminal fluid was analyzed for the concentration of interferon tau (IFNT). Only samples with fully recovered elongating embryos were used for metabolomics analysis (CON = 6 and TARG = 6). In addition, blood was collected to determine the concentration of progesterone (P4). The effects of treatment, group, and their interaction on P4 and IFNT were analyzed by ANOVA. Prior to statistical analysis, the concentration of metabolites in embryonic tissues were subjected to loess normalization. Depending on normality, data were analyzed either by ANOVA or Wilcoxon-Mann-Whitney test. The concentration of progesterone and IFNT did not differ between treatments (P = 0.57). The concentration of IFNT in flushing media in CON was 3693.92 ± 5133.24 pg/mL and in TARG was 4132.62 ± 5742.87 pg/mL. There were seven significantly different metabolites (P ≤ 0.05) and 17 tended to be significant (0.05 < P ≤ 0.1) in embryonic tissues at d 16. The metabolites modulated in embryonic tissues belonged to triglyceride, phosphatidylinositol, acylcarnitine, lysphosphatidylglycerol, carboxylic acid, amino acid, phosphatidylethanolamine, phosphatidic acid, diglyceride, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine classes. Among these metabolites, phosphatidylcholines, PC (42:3) and PC (36:5), and one diglyceride were greater in TARG. Meanwhile, phosphatidylinositols, three diglycerides, lysophosphatidylethanolamines, and lysophosphatidylserines were greater in CON. Altogether, the metabolites greater in TARG are related to cell membrane development and lipid metabolism, whereas the metabolites greater in CON are related to structural support in cell membranes, lipid metabolism, and cell-mediated cell signaling. Potential implications for embryo-maternal communication and reproductive outcomes deserve further investigation.

Signaling reprogramming via Stat3 activation unravels high-fidelity human post-implantation embryo modeling.

Learning cell-specific networks from dynamics and geometry of single cells.

A recent study suggested that morphologically diploblastic sea anemones (Cnidaria) have three segregated germ layer identities corresponding to the bilaterian germ layers. Here, we investigated how these germ layer identities are specified during early development of the sea anemone Nematostella vectensis. Our gene expression analysis shows that the mesodermal territory is specified at the animal pole at 6 h postfertilization, followed by the specification of the definitive endoderm between mesoderm and ectoderm. We then assessed the role of β-catenin, MAPK, and Notch signaling during mesoderm and endoderm formation. We show that the mesodermal marker genes are activated by MAPK signaling while being repressed elsewhere by β-catenin signaling. Delta-expressing mesoderm then signals to Notch-expressing ectoderm, inducing the definitive endoderm domain at the mesoderm/ectoderm interface. Gain- and loss-of-function experiments showed that Notch signaling is sufficient for endoderm induction. Based on our results, we propose a model of germ layer specification in Nematostella defined by a crosstalk of MAPK, β-catenin, and Notch signaling. Given the similarity of the germ layer specification between the sea anemone and echinoderms, we propose that triploblasty may have predated the split of cnidarians and bilaterians.

Although gastrulation is a conserved morphogenetic process in animals, the cellular mechanisms underlying it are highly variable. This raises important questions regarding the extent to which conserved or divergent gene regulatory programs (GRNs) control gastrulation in phylogenetically distant taxa. We compared gene expression profiles during gastrulation of Acropora digitifera and Acropora tenuis, species that diverged ∼50 million years ago. Despite the morphological similarity, each species uses divergent GRNs, supporting the concept of developmental system drift. Consistently, orthologous genes showed significant temporal and modular expression divergence, indicating GRN diversification rather than conservation. Yet, we identified a subset of 370 differentially expressed genes that were up-regulated at the gastrula stage in both species, with roles in axis specification, endoderm formation, and neurogenesis, suggesting a conserved regulatory "kernel" for the process. We also identified species-specific differences in paralog usage and alternative splicing patterns that indicate independent peripheral rewiring of this conserved module. Interestingly, although A. digitifera exhibits greater paralog divergence, consistent with neofunctionalization, A. tenuis shows more redundant expression, suggesting the regulatory robustness of developmental programs in this species.

ABSTRACTLactate has been widely recognised as an energy source and metabolic by‐product, but increasing evidence supports its critical role as a signalling molecule or epigenetic substrate. During early embryogenesis, lactate production increases during the transition from early to late blastocyst, coinciding with the differentiation of inner mass cell (ICM) into epiblast (EPI) and primitive endoderm (PrE), termed the second cell fate decision. However, the role of this hallmark metabolic change in the second cell fate segregation remains unknown. Herein, using in vitro and in vivo models, we found lactate production is preferentially increased in PrE cells and is essential for ICM differentiation into PrE. Mechanically, increased lactate in PrE precursor cells and FGF signalling in EPI precursor cells reciprocally activate each other and synergise to prompt PrE specification, forming an intercellular positive feedback loop essential for this lineage commitment. Additionally, lactate enhanced histone lactylation levels during differentiation into PrE fate. Thus, our findings construct a complex multilayer model in which intracellular metabolite in PrE cooperates with intercellular growth factor signalling from EPI to regulate early embryonic lineage commitment. Highlighting the multifaceted lactate's function, our findings also advance the current knowledge that bridges epigenetic reprogramming and metabolic remodelling during early embryonic development.

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signalling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are crucial mediators of FGF signalling in cell lineage specification and maturation in mouse embryos. We show that loss of Etv5 compromises primitive endoderm formation at pre-implantation stages. Furthermore, Etv4 and Etv5 (Etv4/5) deficiency delays naïve pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, Etv4/Etv5-deficient embryos exhibit anterior visceral endoderm migration defects post-implantation, a process essential for coordinated embryonic patterning and gastrulation initiation. Our results demonstrate the successive roles of these FGF signalling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development.

In this study, we demonstrate that cytoskeletal state at the onset of directed differentiation is critical for the specification of human pluripotent stem cells (hPSCs) to all three germ layers. In particular, a polymerized actin cytoskeleton facilitates directed ectoderm differentiation, while depolymerizing F-actin promotes mesendoderm lineages. Applying this concept to a stem cell-derived islet (SC-islet) differentiation protocol, we show that depolymerizing F-actin with latrunculin A (latA) during the first 24 hours of definitive endoderm formation facilitates rapid exit from pluripotency and alters Activin/Nodal, BMP, JNK-JUN, and WNT pathway signaling dynamics. These signaling changes influence downstream patterning of the gut tube, leading to improved pancreatic progenitor identity and decreased expression of markers associated with other endodermal lineages. Continued differentiation generates islets containing a higher percentage of β cells that exhibit improved maturation, insulin secretion, and ability to reverse hyperglycemia. Furthermore, this latA treatment reduces enterochromaffin cells in the final cell population and corrects differentiations from hPSC lines that otherwise fail to consistently produce pancreatic islets, highlighting the importance of cytoskeletal signaling at the onset of directed differentiation.

Vertebrate embryogenesis requires the precisely timed specification of 3 germ cell layers- ectoderm, mesoderm, and endoderm- which give rise to tissues and organs in the developing organism. The tumor suppressor gene NF2, moesin-ezrin-radixin like (MERLIN) tumor suppressor (Nf2) is expressed in all 3 germ layers during mouse development and its homozygous deletion causes embryonic lethality. People with heterozygous NF2 mutations typically develop Schwann cell tumors, especially vestibular schwannoma, but the specific role of NF2 in human embryonic development is unclear. Here, human induced pluripotent stem cells (hiPSCs) are used to demonstrate that NF2 is essential for endoderm specification and formation in humans. Although endoderm differentiation is not impaired in hiPSCs with heterozygous NF2 mutation, NF2 knockout (NF2-/-) abolished the capacity to form endoderm in vitro, confirmed by loss of expression of endoderm-related genes and proteins, or teratomas in vivo. This defect is mediated by the nuclear translocation of yes-associated protein 1 (YAP1), a transcription co-activator regulating lineage fate via the Hippo pathway and subsequent YAP1-mediated shutdown of Activin/Nodal signaling. Endoderm formation can be rescued via YAP1 knockdown or forced re-expression of NF2 in NF2-/- cells. Taken together, the essential role of NF2 during endoderm specification in human embryogenesis as a regulator of YAP1 is reported.

Sox17 is a key transcriptional regulator of endoderm formation and function in the gallbladder, blood vessels and reproductive organs. Although multiple transcript variants of Sox17 have been suggested, the precise mechanisms underlying their time- and tissue-specific expression remain unclear. In this study, we discovered two putative regulatory sequences (R1 and R2) adjacent to different transcription start sites of mouse Sox17 exon 1 and generated deletion mice for these regions (Sox17Δdr/Δdr). Sox17Δdr/Δdr mice were alive and fertile, and they possessed a normal-sized gallbladder. However, semiquantitative analysis of immunostaining showed that the expression levels of SOX17 in Sox17Δdr/Δdr embryos were reduced to less than 50% of the wild-type in the gallbladder epithelium. Furthermore, the bile ductal epithelium marker SOX9 was abnormally upregulated, and PAS/DBA-positive mucin secretion-like epithelial cells were induced in the Sox17Δdr/Δdr gallbladder. Our results demonstrate that the distal sequence of Sox17, including R1 and R2, is important for the regulation of Sox17 gene expression in the embryonic gallbladder and is crucial for normal gallbladder epithelial development.

About 70% of human cleavage stage embryos show chromosomal mosaicism, falling to 20% in blastocysts. Chromosomally mosaic human blastocysts can implant and lead to healthy new-borns with normal karyotypes. Studies in mouse embryos and human gastruloids showed that aneuploid cells are eliminated from the epiblast by p53-mediated apoptosis while being tolerated in the trophectoderm. These observations suggest a selective loss of aneuploid cells from human embryos, but the underlying mechanisms are not yet fully understood. Here, we investigated the cellular consequences of aneuploidy in a total of 125 human blastocysts. RNA-sequencing of trophectoderm cells showed activated p53 pathway and apoptosis proportionate to the level of chromosomal imbalance. Immunostaining corroborated that aneuploidy triggers proteotoxic stress, autophagy, p53-signaling, and apoptosis independent from DNA damage. Total cell numbers were lower in aneuploid embryos, due to a decline both in trophectoderm and in epiblast/primitive endoderm cell numbers. While lower cell numbers in trophectoderm may be attributed to apoptosis, aneuploidy impaired the second lineage segregation, particularly primitive endoderm formation. This might be reinforced by retention of NANOG. Our findings might explain why fully aneuploid embryos fail to further develop and we hypothesize that the same mechanisms lead to the removal of aneuploid cells from mosaic embryos.

About 70% of human cleavage stage embryos show chromosomal mosaicism, falling to 20% in blastocysts. Chromosomally mosaic human blastocysts can implant and lead to healthy new-borns with normal karyotypes. Studies in mouse embryos and human gastruloids showed that aneuploid cells are eliminated from the epiblast by p53-mediated apoptosis while being tolerated in the trophectoderm. These observations suggest a selective loss of aneuploid cells from human embryos, but the underlying mechanisms are not yet fully understood. Here, we investigated the cellular consequences of aneuploidy in a total of 125 human blastocysts. RNA-sequencing of trophectoderm cells showed activated p53 pathway and apoptosis proportionate to the level of chromosomal imbalance. Immunostaining corroborated that aneuploidy triggers proteotoxic stress, autophagy, p53-signaling, and apoptosis independent from DNA damage. Total cell numbers were lower in aneuploid embryos, due to a decline both in trophectoderm and in epiblast/primitive endoderm cell numbers. While lower cell numbers in trophectoderm may be attributed to apoptosis, aneuploidy impaired the second lineage segregation, particularly primitive endoderm formation. This might be reinforced by retention of NANOG. Our findings might explain why fully aneuploid embryos fail to further develop and we hypothesize that the same mechanisms lead to the removal of aneuploid cells from mosaic embryos.

Bone morphogenetic protein signaling pathway– Ethanol interactions disrupt palate formation independent of gata3

Endoderm, one of three primary germ layers of vertebrate embryos, makes major contributions to the respiratory and gastrointestinal tracts and associated organs, including liver and pancreas. In mammals, the transcription factor SOX17 is vital for endoderm organ formation and can induce endoderm progenitor identity. Duplication of ancestral sox17 in the teleost lineage produced the paralogues sox32 and sox17 in zebrafish. Sox32 is required for specification of endoderm and progenitors of the left-right organiser (Kupffer's Vesicle, KV), with Sox17 a downstream target of Sox32 that is implicated in further KV development. Phenotypic evidence therefore suggests functional similarities between zebrafish Sox32 and Sox17 and mammalian SOX17. Here, we directly compare these orthologues and paralogues, using the early zebrafish embryo as a biological platform for functional testing. Our results indicate that, unlike Sox32, human SOX17 cannot induce endoderm specification in zebrafish. Furthermore, using hybrid protein functional analyses, we show that Sox32 specificity for the endoderm gene regulatory network is linked to evolutionary divergence in its DNA-binding HMG domain from its paralogue Sox17. Additionally, changes in the C-terminal regions of Sox32 and Sox17 underpin their differing target specificities. Finally, we establish that specific conserved peptides in the C-terminal domain are essential for the role of Sox17 in establishing correct organ asymmetry. Overall, our results illuminate the molecular basis for functional divergence of Sox32 and Sox17 in vertebrate endoderm development and left-right patterning, and the evolution of SoxF transcription factor function.

Thyroid gland diseases remain clinical challenges due to the lack of reliable in vitro models to examine molecular pathways of thyrocytes development, maturation, and functional maintenance. This study aimed to develop in vitro thyrocytes model using a stem cell culture, P19 embryonal carcinoma which requires no feeder layer, differentiation into mature and functional thyrocytes that allow molecular and genetic manipulation for studying thyroid diseases. The procedure utilizes Activin A and thyroid stimulating hormone (TSH) to first induce embryoid body endoderm formation enriched in thyrocyte progenitors. Following dissociating embryoid bodies, thyrocyte progenitors are plated in Matrigel as monolayer cultures that allows thyrocyte progenitors mature to functional thyrocytes. These thyrocytes further maturate to form follicle-like structures expressing and accumulating thyroglobulin that can be secreted into the medium upon TSH stimulation. Thyrocyte differentiation-maturation process is monitored by the expression of essential transcriptional factors and thyrocyte-specific functional genes. Further, the applicability of this system is validated by introducing a siRNA control. Following molecular manipulation, the system can still be guided to differentiate into mature and functional thyrocytes. This system spans a time frame of 14 days, suitable for detailed molecular studies to dissect pathways and molecular players in thyrocytes development and functional maintenance.

Deciphering the underlying gene regulatory networks (GRNs) that govern early human embryogenesis is critical for understanding developmental mechanisms yet remains challenging due to limited sample availability and the inherent complexity of the biological processes involved. To address this, we developed InPheRNo-ChIP, a computational framework that integrates multimodal data, including RNA-seq, transcription factor (TF)-specific ChIP-seq, and phenotypic labels, to reconstruct phenotype-relevant GRNs associated with endoderm development. The core of this method is a probabilistic graphical model that models the simultaneous effect of TFs on their putative target genes to influence a particular phenotypic outcome. Unlike the majority of existing GRN inference methods that are agnostic to the phenotypic outcomes, InPheRNo-ChIP directly incorporates phenotypic information during GRN inference, enabling the distinction between lineage-specific and general regulatory interactions. We integrated data from three experimental studies and applied InPheRNo-ChIP to infer the GRN governing the differentiation of human embryonic stem cells into definitive endoderm. Benchmarking against a scRNA-seq CRISPRi study demonstrated InPheRNo-ChIP's ability to identify regulatory interactions involving endoderm markers FOXA2, SMAD2, and SOX17, outperforming other methods. This highlights the importance of incorporating the phenotypic context during network inference. Furthermore, an ablation study confirms the synergistic contribution of ChIP-seq, RNA-seq, and phenotypic data, highlighting the value of multimodal integration for accurate phenotype-relevant GRN reconstruction.

Genome-wide association study of growth and reproductive traits based on low-coverage whole-genome sequencing in a Chubao black-head goat population

N6-methyladenonsine (m6A) is ubiquitously distributed in mammalian mRNA. However, the precise involvement of m6A in early development has yet to be fully elucidated. Here, we report that deletion of the m6A demethylase ALKBH5 in human embryonic stem cells (hESCs) severely impairs definitive endoderm (DE) differentiation. ALKBH5−/− hESCs fail to undergo the primitive streak (PS) intermediate transition that precedes endoderm specification. Mechanistically, we show that ALKBH5 deficiency induces m6A hypermethylation around the 3′ untranslated region (3′UTR) of GATA6 transcripts and destabilizes GATA6 mRNA in a YTHDF2-dependent manner. Moreover, GATA6 binds to the promoters of critical regulatory genes involved in Wnt/β-catenin signaling transduction, including the canonical Wnt antagonist DKK1 and DKK4, which are unexpectedly repressed upon the dysregulation of GATA6 mRNA metabolism. Remarkably, DKK1 and DKK4 both exhibit a pleiotropic effect in modulating the Wnt/β-catenin cascade and guard the endogenous signaling activation underlying DE formation as potential downstream targets of the ALKBH5-GATA6 regulation. Here, we unravel a role of ALKBH5 in human endoderm formation in vitro by modulating the canonical Wnt signaling logic through the previously unrecognized functions of DKK1/4, thus capturing a more comprehensive role of m6A in early human embryogenesis.

BackgroundThe 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.ResultsHere 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.ConclusionsBy 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.