Frontiers in Cell and Developmental Biology

Somatic cell nuclear transfer (SCNT) remains inefficient, limiting its practical use in cattle reproduction and research. This study investigated two complementary strategies to enhance handmade cloning (HMC): (1) holding bovine oocytes overnight and delaying maturation to enable a second round of SCNT and (2) using bovine-induced pluripotent stem cells (biPSCs) as donor nuclei to enhance developmental competence. Bovine oocytes were subjected to either conventional in vitro maturation (CONV; 20 h) or delayed maturation using a holding medium for 20 h before CONV (HOLD). Matured oocytes were used for SCNT, parthenogenetic activation (PA), or in vitro fertilization (IVF) as controls. Handmade SCNT embryos were reconstructed using fibroblasts or biPSCs as donors, activated, and cultured for 7 days. Results showed no significant differences between CONV and HOLD groups in oocyte maturation, recovery after stripping, survival after zona removal, or cleavage and blastocyst development after SCNT. Fusion rates using fibroblasts were comparable between groups (42.6{+/-}6.0% vs. 50.3{+/-}9.8%), with biPSCs showing significantly higher fusion rates in CONV group (85.7{+/-}8.2% vs. 50.5{+/-}8.8%, P<0.05). Among fused embryos, biPSCs produced higher blastocyst rates (33.3{+/-}16.7%) compared with fibroblast donors (21.9{+/-}12.6%, P<0.05). Across all reconstructed embryos, cleavage and blastocyst development were also greater with biPSCs (odds ratios 3.4 and 2.7 respectively). These findings indicate that delaying maturation offers flexible timing for SCNT without compromising competence. Moreover, biPSCs enhance embryo developmental outcomes, supporting their use as superior donor cells for advancing cloning efficiency and applications in reproductive biotechnology.

Embryo implantation requires execution of highly synchronized processes at the feto-maternal interface, initiated by blastocyst attachment to the endometrial epithelium. Hyaluronan is a major ECM component known to regulate adhesion-associated biological processes in various physiological settings. We hypothesized that hyaluronan may facilitate blastocyst attachment. In order to test our hypothesis, we characterized the blastocyst expression of hyaluronan synthesizing and degrading enzymes, as well as the expression of hyaluronan receptors during attachment. The functional impact of hyaluronan was challenged by the use of mouse transgenic blastocysts, in which genes encoding for hyaluronan synthesizing enzymes were deleted using lentiviral incorporation of Cas-9 endonuclease alongside specific short-guide RNAs into the embryonic trophectoderm. Embryos with transgenic trophectoderm were tested for their attachment in vitro, or assessed for implantation in vivo, upon transfer to foster dams. Deletion of the trophectoderm hyaluronan biosynthesis significantly reduced the number of blastocysts attached to human uterine epithelium cells in vitro. Reduced attachment was also observed in vivo, in pregnant mice carrying blastocysts with hyaluronan-depleted trophectoderm. In agreement, trophectoderm expression of osteopontin, was downregulated upon depletion of hyaluronan. MRI measurements revealed a decrease in uterine blood vessels permeability. Uterine expression of VEGF-A, PTGS-2 and uterine osteopontin, which constitute the immediate response to blastocyst attachment was also reduced. Furthermore, impaired implantation, associated with a decrease in hyaluronan synthesis in the mural trophectoderm, obtained upon tamoxifen treatment, has been recovered by LIF administration. These results demonstrate that estrogen-regulated hyaluronan-synthesis in the trophectoderm is indispensable for mouse blastocysts attachment to the uterine epithelium.

Here we describe a novel neuro-mesodermal assembloid model which recapitulates aspects of peripheral nervous system (PNS) development such as neural crest cell (NCC) induction, delamination, migration and sensory as well as sympathetic ganglion formation. The ganglia send neuronal projections to the mesodermal as well as the neural compartment. Axons in the mesodermal part are associated with Schwann cells. In addition, peripheral ganglia as well as nerve fibers interact with the co-developing vascular plexus, forming a neurovascular niche. Finally, developing sensory ganglia show response to capsaicin treatment indicating their functionality. The presented assembloid model could help to uncover mechanisms of NCC delamination, migration and PNS development in the human tissue context. Moreover, the model could be used for toxicity screenings or drug testing. The co-development of mesodermal and neuroectodermal tissues and of a well-organized vascular plexus along with a peripheral nervous system allows to investigate the crosstalk between neuroectoderm and mesoderm and between peripheral neurons/neuroblasts and endothelial cells. Such interactions influence NCC delamination and migration, sensory neuron differentiation and rearrangement of the primitive vascular plexus in the embryo.

Transforming growth factor-beta (TGF-{beta}) supports the in vitro maintenance of embryonic and trophoblast stem cells. Here, we demonstrated that, in a sheep embryo model, the transition from morula to blastocyst is positively regulated by TGF-{beta}3, primarily through its promotion of trophoblast development. Our results indicate that morulae treated with TGF-{beta}3 develop at a higher rate into blastocysts, characterized by an expanded trophoblast layer marked by CDX-2 expression. In blastocysts, TGF-{beta}3 mediates transcriptional activation of genes involved in cell adhesion and lipid metabolism pathways, leading to remarkable in vitro outgrowth expansion and a substantial increase in trophoblast lipid droplet content. Functional analysis reveal that the positive effects of TGF-{beta}3 are mitigated by inhibition of Acetyl-CoA Synthetase Short-Chain Family Member 2 (ACSS2), a key enzyme upregulated by TGF-{beta}3 and a promoter of de novo lipgenensis. These findings suggest that TGF-{beta}3 modulates lipid metabolism during blastocyst formation and may play a potential role in regulating implantation and placental development.

Primordial follicle assembly in mammals occurs at perinatal ages and largely determines the ovarian reserve available to support the reproductive lifespan. The primordial follicle structure is generated by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time-series on mouse ovaries coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to PD3 are reported for the first time. The time frame of analysis comprises the breakdown of germ cell cysts and the assembly of primordial follicles. Confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified ten distinct gene clusters associated to germ cells and eight to granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. Building single-cell pseudo temporal trajectories five states and two branch points of fate transition for the germ cells, and three states and one branch point for the granulosa cells were revealed. Moreover, GO and ClueGO term enrichment enabled identifying biological processes, molecular functions and cellular components more represented in germ cells and granulosa cells or common to both cell types at each specific stage. Finally, by SCENIC algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell specific transcription programs throughout the investigated period.

Somatic cell reprogramming in vitro prior to nuclear transfer is one strategy expected to improve clone survival during development. In this study, we investigated the reprogramming extent of fish fin somatic cells after in vitro exposure to Xenopus egg extract and subsequent culture. Using a cDNA microarray approach, we observed drastic changes in the gene expression profile of the treated cells. Several actors of the TGF{beta} and Wnt/{beta}-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove somatic cells towards a mesenchymal-epithelial transition (MET), the hallmark of somatic reprogramming in induced pluripotent stem cells (iPSCs). However, treated cells were also characterized by a strong decrease in de novo lipid biosynthesis metabolism, the lack of re-expression of pou2 and nanog pluripotency markers, and absence of DNA methylation remodeling of their promoter region. In all, this study showed that Xenopus egg extract treatment initiated an in vitro reprogramming of fin somatic cells in culture. Although not thorough, the induced changes have primed the somatic chromatin for a better embryonic reprogramming upon nuclear transfer.

The zona pellucida (ZP) is the quintessential extracellular structure of mammalian oocytes. Contrary to long-standing view that the synthesis of ZP proteins is specific to oocytes and muted in embryos, we report here that the major zona pellucida protein ZP2 is re-synthesized and functionally required during mouse embryo development. The orthogonal methods of mass spectrometry and monoclonal immunofluorescence revealed an increase of ZP2 abundance at the 8-cell / morula stage, which did not occur when zygotes were microinjected with translation-blocking oligonucleotides (morpholinos). To shed light on the functional significance of embryonic ZP2, we performed protein knockdown using immunodepletion (by Trim-Away) while at the same time preventing replenishment (by translation-blocking morpholino). ZP2 knockdown resulted in morula stage retardation and formation of defective blastocysts, whose cell lineages trophectoderm and primitive endoderm were smaller and less able to support post-implantation development. The transcriptional correlates of these morphological alterations had a gene ontology (biological process) signature that included cell lineage-relevant terms ( endoderm development, gastrulation), while the proteomic correlates had a gene ontology signature related to protein synthesis. Taken together, these results call into question the traditional model that ZP proteins function solely in the extracellular space and accompany embryogenesis as passive bystanders: on the contrary, ZP proteins also participate actively in the intracellular processes of early embryogenesis.

Actin-related proteins (Arps) are a superfamily of proteins which share sequence similarities with conventional actin and are involved in different cellular processes. Actin-related protein M1 (ARPM1) also known as actin-related protein T3 (ACTRT3) is a testis-enriched Arp which can be found in the perinuclear theca (PT) of murine round and elongating spermatids. ARPM1 forming a complex with Profilin 3 (PFN3) is lost in Pfn3-deficient sperm. We generated a mouse model deficient for Arpm1 and demonstrate that Arpm1-/- male mice are subfertile, with morphological aberrations of the acrosome. During spermiogenesis, defects become apparent in Cap phase of acrosome biogenesis when abnormal acrosomal granules are observed. Arpm1-deficiency causes deregulation of GM130 and TGN46 suggestive of defects in cis- and trans-Golgi trafficking required for acrosome development. Co-immunoprecipitation revealed that ARPM1 interacts with the PT-specific proteins ACTRT1, ACTRT2, ACTL7A and the sperm surface protein ZPBP, additionally to its already shown interaction with PFN3. We propose that ARPM1 acts as a structural component of the PT contributing to the cytoskeletal network connecting acrosome and nucleus. In addition, ARPM1 mediates the localization of ZPBP to enable fertilization and it tethers PFN3 to properly regulate Golgi-related acrosome development.

To achieve a stereotypic lineage, each embryo of Caenorhabditis elegans follows an invariant cell differentiation process arising from a combination of cell polarisation, asymmetric or symmetric divisions, combined with intercellular signalling processes. This pattern of embryonic cell differentiation is driven by regulated segregation of molecules occurring at each cell division, including polarity proteins or cell fate determinants, transcription factors, p-granules and mRNAs. These distribution patterns are coupled with a robust spatio-temporal orchestration of cortical actin dynamics, which also plays a crucial role in these processes. However, compared to other molecular contents, how the actin per se is segregated from the first asymmetric division onward remains poorly understood. This study presents a thorough quantification of the intracellular distribution from the zygote to the 4-cell stage of key actors related to actin polymerisation: two nucleators (a formin and the Arp2/3 complex), a capping protein and E-cadherin. We additionally developed a novel method to assess actin polymerisation capacities from single blastomere extracts. We found that actin-related signatures arise at these early stages and that differential mechanisms of protein segregation and homeostasis occur, depending both on the cell pair and on the protein considered. Notably, if asymmetric divisions correlated with unequal partitioning of actin-related contents in a process linked with embryonic polarity, differences were revealed between AB daughter cells upon their separation. Taken together, these actin-related asymmetric distributions are adding a layer to the complexity of cell fate acquisition mechanisms in the early embryo.

Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied, however, most of the studies relates to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells demonstrates limited success. Here, we describe a simple, efficient and reproductive protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when placed in the interaction with the primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures and the mESC-derived UB cells formed network of collecting ducts, connected with the nephron tubules. Altogether, our studies established an uncomplicated and reproducible platform for kidney disease modelling, drug testing and regenerative medicine applications.

Extracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarian Schmidtea mediterranea had similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up more quickly by S/G2 neoblasts than G1 neoblasts/early progeny and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced upregulation of neoblast-associated transcripts. In addition, EV injection increased the number of F-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenable in vivo model for the study of EV function during regeneration.

While significant progress has been made in understanding different aspects of liver regeneration, the molecular mechanisms responsible for the initiation and termination of cell proliferation in the liver after massive loss or injury of liver tissue remain unknown. The loss of liver mass affects tissue-specific mitogenic inhibitors in the blood, which in turn regulate the proliferation of remaining hepatocytes and liver regeneration. Although well described in a number of publications, which inhibitory substances or "sensor molecules" control the regeneration mechanisms to properly maintain liver size remain unknown. Extracellular vesicles (EVs) are nano-sized, membrane-limited structures secreted by cells into the extracellular space. Their proposed role is stable intercellular carriers of proteins and RNAs, mostly micro-RNA, from secreted to recipient cells. Taken up by the recipient cells, EVs can significantly modulate their biological functions. In the present study, using in vivo and in vitro models, we demonstrate that hepatocyte proliferation and liver regeneration are regulated by EVs secreted by hepatocytes into the bloodstream. This regulation is carried out through a negative feedback mechanism, which explains the very precise regeneration of liver tissue after massive damage. We also demonstrate that an essential component of this mechanism is RNA carried by hepatocyte-derived EVs. These findings open up a new and unexplored area of biology regarding the mechanisms involved in the homeostasis regulation of various constantly renewing tissues by maintaining the optimal size and correct ratio between differentiating and proliferating cells.

In mammalian females, primordial follicles form during fetal ovarian development and serve as the only source to sustain adult ovarian function. Mechanisms underlying how primordial follicles assemble, maintain dormancy, activate for follicular development, and undergo cell death are important for understanding ovarian physiology and pathological conditions. Here, we demonstrate a protocol of culturing postnatal mouse ovaries on membrane inserts - an approach allowing culture, pharmaceutical treatment, and live-imaging of intact ovaries for up to 10 days depending on the developmental stage of the ovary. The change of culture conditions can be done by transferring inserts containing cultured ovaries between wells on a plate, avoiding physical interference with tissues during culture. In this experiment, we use postnatal day 5 (P5) CD1 mouse ovary culture as an example. P5 ovaries were isolated and placed on a 12 mm insert in a 24-well plate. Each ovary was separated within a droplet of DMEM/F12 medium supplemented with 10% FBS, 3 mg/ml BSA, 10 mIU/ml FSH, and Gibco Antibiotic-Antimycotic, and gently stabilized to the membrane insert. The medium was changed every two days, and the culture was maintained for five days. Following the culture, ovaries were fixed in 4% paraformaldehyde for two hours and processed for whole-mount antibody staining. Primordial follicles were visualized using confocal microscopy with anti-DDX4 antibody, allowing for the analysis of oocyte number and morphology. We showed that the number of primordial follicles in each ovary was significantly affected by whether tissues were properly placed on the membrane insert. Difference in the number of ovaries on each insert may contribute to non-biological variations and should be avoided.

In vitro fertilization (IVF) is key for genetic improvement programs in bovine. However, embryos produced through IVF have lower developmental competence than those produced under in vivo conditions. Conventional sperm preparation for IVF typically relies on heparin for sperm capacitation but fails to replicate the finely tuned molecular environment of the oviduct, resulting in compromised embryonic competence. Here, we evaluated the effect of HyperBull, a novel capacitation technology, on bovine IVF outcomes using unsorted cryopreserved semen. In a split-sample design, 528 cumulus-oocyte complexes were co-incubated with either control or HyperBull capacitated spermatozoa from the same bull. While overall blastocyst rates were not significantly different between groups (34.21% HyperBull vs. 28.63% control, p=0.148), the proportion of hatched embryos was significantly higher in the HyperBull group (15.82% vs. 9.13%, p=0.016). These findings suggest that modulating capacitation signals prior to insemination enhances embryonic developmental competence, thereby improving readiness for implantation. HyperBull may thus represent a valuable tool to increase the efficiency of IVF programs.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

Dietary and addictive small molecules play a significant role in altering in vivo conditions. Due to their minuscule size, these molecules can seamlessly traverse tissues and cellular membranes, influencing key biological processes such as cellular growth, differentiation, and intracellular communication, which are crucial for tissue regeneration. The zebrafish (Danio rerio) serves as an excellent model for studying regenerative growth due to its remarkable ability to regrow amputated appendages. In this study, we systematically evaluated the effect of small molecules, including ethanol (0.5%), glucose (1%), and NaCl (0.2%), on zebrafish caudal fin regeneration over a 7-day period. Regenerative growth analysis indicated delayed fin regrowth across all treated groups, with ethanol exposure showing the most significant impairment. Behavioural assessments revealed significant stress-induced locomotor alterations in treated groups, with the ethanol-exposed group exhibiting the most pronounced reduction in total distance moved and velocity. Proteomic profiling using label-free quantification (LFQ) identified 113, 257, and 178 differentially expressed proteins in ethanol, glucose, and NaCl-treated groups, respectively. Subsequent validation using the iTRAQ labeling approach confirmed 16 commonly dysregulated proteins across all conditions, highlighting a shared molecular response associated with stress and repair mechanisms. Pathway enrichment analysis mapped differentially expressed proteins to various canonical signaling pathways, including GP6 signaling, mitochondrial dysfunction, RHO GTPase cycling, antigen processing, and metabolic regulation. Ingenuity Pathway Analysis (IPA) further revealed associations with disease and function networks specific to each treatment condition. Our findings provide valuable insights into how metabolic and ionic perturbations influence zebrafish fin regeneration at the molecular level, offering a deeper understanding of tissue repair mechanisms under stressed conditions.

Sex development is an intricate and crucial process in all vertebrates that ensures the continued propagation of genetic diversity within a species, and ultimately their survival. Perturbations in this process can manifest as variations/differences of sex development (VSD/DSD). Primary gonadal somatic cells - the ovarian granulosa cells (GCs) in the case of women - represent the absolute model to investigate mechanism of disease in VSDs. Collection of these cells in humans is laborious and invasive, while classical animal models fail to recapitulate the human phenotype and function. Furthermore, in patients with the most severe forms of VSD gonadal cells are totally absent. It is therefore vital to develop an alternative cell-model. In view of this, we established an efficient method to reprogram donor-derived urinary progenitor cells (UPs) and differentiate iPSCs into granulosa-like cells (GLCs). The UPs presented a less invasive and high-quality cell source, improving the clinical applicability of the model along with utilising a non-integrative reprogramming method that eliminates alteration of the original genome. This novel GLC model closely resembles human GCs in morphology and marker gene expression of GC cell-fate and essential function. These results provide the prospect to generate patient-specific personalised GC models to investigate mechanism of disease in VSDs and could improve understanding of the intricacies in female gonadal development.

Alanine tRNAs (UGC) control the development of the innate and the environment-modulated acquired C. elegans chemo-attractive responses. Some Ala-tRNA isomers are required for the development of the chemo-attractive behavior (dev-tRNAs), while others (odor-tRNAs) are made as life-term olfactory imprints of early larval odor-exposures. dev-tRNAs and odor-tRNAs biosynthesis respectively require the tRNA modifying Elongator complex sub-units ELPC-3 and ELPC-1: while elpc-3 mutants are chemo-attraction deficients, elpc-1 mutants do not synthesize odor-tRNAs imprints. Feeding wild-type dev-tRNAs restore a wild-type behavior in elpc-3 mutants. Feeding purified odor-tRNAs enhances odor responses (positive imprinting) in adult wild-type worms, while it decreases odor responses (negative imprinting) in adult imprinting deficient elpc-1 mutants. Both positive and negative imprinting can be stably inherited in worm populations. Crossing experiments indicate that both behavioral phenotypes segregate as monogenic monoallelic alterations, following Mendelian inheritance rules. Co-culture and food conditioning suggest the developmental and the odor-specific regulatory Ala-tRNAs are released in worms environment. Commensal naive acquire odor-specific imprinting from odor-experienced, while co-culture together with wild-type animals fully rescues the chemo-attractive defects of the elpc-3 mutants. Worm to worm communication of imprinting require a number of RNA interference (RNAi) genes as the intestinal RNA transporter SID-2, the initial exogenous RNAi Dicer/RDE-1/DRH-1-2/RDE-4 complexe, and the RNA-dependent RNA polymerase RRF-3. Moreover, a male contribution of the 3-exonuclease ERI-1 activity determines whether olfactory imprints will be erased or stably fixed and inherited in worms progeny. The RNAi processing of externalized chemosensory regulatory Ala-tRNAs would generate small interfering tRNAs (si-tRNAs) able to target only tRNA complementary sequences present on worm genome, that is the tDNA genes and the transcription independent extra-TFIIIC sites. A model of control loop in which olfactory receptor expression levels in chemosensory neurons could be non-genetically but stably regulated via RNAi processing of secreted constitutive or environment-modified Ala-tRNAs is discussed.

The basal lamina is a specialized sheet of dense extracellular matrix (ECM), linked to the plasma membrane of specific cell types in their tissue context, that serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies, and specific types of brain malformation. The latter three pathologies occur in the dystroglycanopathies, which are caused by dysfunction of the ECM receptor dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted due to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells to model basal lamina formation. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy revealed ultrastructural basal lamina defects and reduced ECM assembly. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in muscular dystrophies. Finally, we applied this system to evaluate an experimental ribitol supplement therapy on genetically diverse dystroglycanopathy patient samples.

During mouse embryogenesis, the interactions between the epiblast and extraembryonic endoderm are critical for germ layer specification and body plan development. Gastruloids recapitulate aspects of gastrulation in the absence of morphogenic signals from the extraembryonic environment. This favors a predominantly posteriorized and dorsalized phenotype with a limited representation of embryonic lineages. Here, we develop and employ a co-aggregation (aggregoid) approach combining embryonic and extraembryonic endoderm-like cells to mimic spatial interactions in developing mouse embryos. The obtained embryo models show the appearance of node and notochord, enriched endoderm populations, increased mesoderm diversity including cardiopharyngeal lineages and vascular endothelium in an overall anteriorized and ventralized phenotype. The work delineates a versatile strategy for refining stem cell-based embryo models to achieve specific morphotypes.