Biology subjects

Zernicka-Goetz, M.

Publications and source records attributed to Zernicka-Goetz, M..

14 recordsLinked to original sources

bioRxiv2026

Lineage tracing reveals asynchronous fate restriction in mouse embryos and stem cell-derived embryo models

How a flexible mammalian embryo gives rise to a reproducible body plan remains unclear, partly because cell lineages cannot be followed continuously in vivo. We combined DNA Typewriter lineage recording with tetraploid complementation to reconstruct high-resolution single-cell genealogies across mouse development and built FateVec, an analysis framework that infers fate-bias dynamics and lineage-associated transcriptional programs from reconstructed phylogenies. Loss of developmental potential proved progressive and asynchronous, with multiple lineages acquiring detectable fate bias before morphological diversification at gastrulation. Within the neural crest, most cells remained multipotent over an extended window, while independent founder clades differed markedly in restriction timing. Extending lineage recording to a stem-cell-derived embryo model revealed that cells with shared transcriptional identities can arise from distinct lineage relationships. We further found embryonic-lineage contributions to extraembryonic-like states, exposing an unexplored lineage plasticity. This lineage-resolved view of embryogenesis reveals that cell identity alone predicts neither developmental ancestry nor the timing of fate restriction.

developmental biology↗

bioRxiv2025

Spindle-localized F-actin regulates polar MTOC organization and the fidelity of meiotic spindle formation

Mammalian oocytes are notoriously prone to chromosome segregation errors leading to aneuploidy. The spindle provides the machinery for accurate chromosome segregation during cell division. Mammalian oocytes lack centrioles and, therefore, the meiotic spindle relies on the organization of numerous acentriolar microtubule organizing centers into two poles (polar MTOCs, pMTOCs). The traditional view is that, in mammalian oocytes, microtubules are the sole cytoskeletal component responsible for regulating pMTOC organization and spindle assembly. We identified a novel F-actin pool that surrounds pMTOCs, forming F-actin cage-like structure. We demonstrated that F-actin localization on the spindle depends on unconventional myosins X and VIIb. Selective disruption of spindle-localized F-actin, using myosin X/VIIb knockdown oocytes or photoswitchable Optojasp-1, perturbed pMTOC organization, leading to unfocused spindle poles and chromosome missegregation. Here, we unveil an important function of F-actin in regulating pMTOC organization, a critical process for ensuring the fidelity of meiotic spindle formation and proper chromosome segregation.

cell biology↗

bioRxiv2024

Proteome asymmetry in mouse and human embryos before fate specification

Pre-patterning of the embryo, driven by spatially localized factors, is a common feature across several non-mammalian species1-4. However, mammals display regulative development and thus it was thought that blastomeres of the embryo do not show such pre-patterning, contributing randomly to the three lineages of the blastocyst: the epiblast, primitive endoderm and trophectoderm that will generate the new organism, the yolk sac and placenta respectively 4-6. Unexpectedly, early blastomeres of mouse and human embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts7-12. Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Here, by utilizing multiplexed and label-free single-cell proteomics by mass-spectrometry13, we show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins exhibiting strong functional enrichment for protein synthesis, transport, and degradation. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. These protein asymmetries increase at 4-cell stage. Intriguingly, halved mouse zygotes display asymmetric protein abundance that resembles alpha and beta blastomeres, suggesting differential proteome localization already within zygotes. We find that beta blastomeres give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres and that vegetal blastomeres, which are known to have a reduced developmental potential, are more likely to be alpha. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. To our knowledge, this is the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation.

developmental biology↗

bioRxiv2024

Asynchronous mouse embryo polarization leads to heterogeneity in cell fate specification

The first lineage allocation in mouse and human embryos separates the inner cell mass (ICM) from the outer trophectoderm (TE). This symmetry breaking event is executed through polarization of cells at the 8-cell stage and subsequent asymmetric divisions, generating polar (TE) and apolar (ICM) cells. Here, we show that embryo polarization is unexpectedly asynchronous. Cells polarizing at the early and late 8-cell stage have distinct molecular and morphological properties that direct their following lineage specification, with early polarizing cells being biased towards producing the TE lineage. More recent studies have also implicated heterogeneities between cells prior to the 8-cell stage in the first lineage allocation: cells exhibiting reduced methyltransferase CARM1 activity at the 4-cell stage are predisposed towards the TE fate. Here, we demonstrate that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and TE specification. These findings provide a link between asymmetries at the 4-cell stage and polarization at the 8-cell stage, mechanisms of the first lineage allocation that had been considered separate.

developmental biology↗

bioRxiv2023

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos

Human fertility is suboptimal in part by error-prone divisions during early cleavage stages, which frequently result in chromosomal aneuploidy. Most human pre-implantation embryos are mosaics of euploid and aneuploid cells, yet those with a low proportion of aneuploid cells can develop to term at rates similar to fully euploid embryos. How embryos manage aneuploidy during early development remains poorly understood - yet this knowledge is crucial for improving fertility outcomes and reducing developmental defects. To investigate these mechanisms, we established a new mouse model of chromosome mosaicism to trace the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to induce aneuploidy. Here, we demonstrate that the more specific Mps1 inhibitor AZ3146 similarly disrupts chromosome segregation but supports higher developmental potential than reversine. AZ3146-treated embryos transiently upregulate Hypoxia Inducible-Factor-1A (HIF1A) without triggering p53 activation. Given that pre-implantation embryos develop in a hypoxic environment in vivo, we further explored the role of oxygen tension. Hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in mosaic epiblast. Conversely, HIF1A inhibition decreased the proportion of aneuploid cells. Together, these findings uncover a role for hypoxia signaling in modulating the response to chromosomal errors and suggest new strategies to improve the developmental potential of mosaic human embryos.

developmental biology↗

bioRxiv2023

Transgene directed induction of a stem cell-derived human embryo model

The human embryo undergoes morphogenetic transformations following implantation into the uterus, yet our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. Stem cell-derived models of the embryo are important tools to interrogate developmental events and tissue-tissue crosstalk during these stages1. Here, we establish a human post-implantation embryo model comprised of embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cells generated by transcription factor overexpression with wildtype embryonic stem cells and promote their self-organization into structures that mimic aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by hypoblast-and trophoblast-like tissues. We demonstrate that these inducible human embryoids robustly generate several cell types, including amnion, extraembryonic mesenchyme, and primordial germ cell-like cells in response to BMP signaling. This model also allowed us to identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells2. Modulation of the subpopulations in the hypoblast-like compartment demonstrated that extraembryonic-like cells impact epiblast-like domain differentiation, highlighting functional tissue-tissue crosstalk. In conclusion, we present a modular, tractable, integrated3 model of the human embryo that will allow us to probe key questions of human post-implantation development, a critical window when significant numbers of pregnancies fail.

developmental biology↗

bioRxiv2023

spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these unique properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

developmental biology↗

bioRxiv2022

Mouse-embryo model derived exclusively from embryonic stem cells undergo neurulation and heart development

Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extra-embryonic lineages of the post-implantation embryo by transcription factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation, and formation of the anterior-posterior axis, brain, a beating heart structure, and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages, but also showed when and where the model diverges from the natural program. Our findings demonstrate an extra-ordinary plasticity of ESCs to self-organize and generate a whole embryo-like structure.

developmental biology↗

bioRxiv2022

Stem cell-derived mouse embryos develop within an extra-embryonic yolk sac to form anterior brain regions and a beating heart

Embryo-like structures generated from stem cells can achieve varying developmental milestones, but none have been shown to progress through gastrulation, neurulation, and organogenesis.1-7 Here, we show that "ETiX" mouse embryos, established from embryonic stem cells aggregated with trophoblast stem cells and inducible extraembryonic endoderm stem cells, can develop through gastrulation and beyond to undertake neural induction and generate the progenitors needed to create the entire organism. The head-folds of ETiX embryos show anterior expression of Otx2, defining forebrain and midbrain regions that resemble those of the natural mouse embryo. ETiX embryos also develop beating hearts, trunk structures comprising a neural tube and somites, tail buds containing neuromesodermal progenitors and primordial germ cells, and gut tubes derived from definitive endoderm. A fraction of ETiX embryos show neural tube abnormalities, which can be partially rescued by treatment with the metabolically active form of folic acid, reminiscent of common birth defects and therapies in humans. Notably, ETiX embryos also develop a yolk sac with blood islands. Overall, ETiX embryos uniquely recapitulate natural embryos, developing further than any other stem-cell derived model, through multiple post-implantation stages and within extra-embryonic membranes.

developmental biology↗

bioRxiv2021

CADHERIN mediated AMIS localisation

Individual cells within de novo polarising tubes and cavities must integrate their forming apical domains into a centralised apical membrane initiation site (AMIS). This is necessary to enable organised lumen formation within multi-cellular tissue. Despite the well documented importance of cell division in localising the AMIS, we have found a division-independent mechanism of AMIS localisation that relies instead on Cadherin-mediated cell-cell adhesion. Our study of de novo polarising mouse embryonic stem cells (mESCs) cultured in 3D suggest that cell-cell adhesion localises apical proteins such as PAR-6 to a centralised AMIS. Unexpectedly, we also found that mESC cell clusters lacking functional E-cadherin still formed a lumen-like cavity in the absence of AMIS localisation but did so at a later stage of development via a closure mechanism, instead of via hollowing. This work suggests that there are two, interrelated mechanisms of apical polarity localisation: cell adhesion and cell division. Alignment of these mechanisms in space allows for redundancy in the system and ensures the development of a coherent epithelial structure within a growing organ.

developmental biology↗

bioRxiv2021

Reconstructing human early embryogenesis in vitro with pluripotent stem cells

Understanding human development is of fundamental biological and clinical importance. Yet despite its significance, insights into early developmental events in humans still remain largely unknown. While recent advances show that stem cells can mimic embryogenesis1-9 to unravel hidden developmental mechanisms, a stem cell-based model of early human embryogenesis is lacking. Here, we use human extended pluripotent stem cells10to reconstitute early human development in 3-dimensions and recapitulate early embryo-like events. We first perform a systematic characterisation to reveal unique signalling requirements for building the human pre-implantation blastocyst. Further, we show that these in vitro stem cell-derived blastocyst-like structures are able to undertake spatiotemporal self-organisation to mimic peri-implantation remodelling in which a polarised rosette opens up the amniotic cavity within a developing disc. The hallmarks of human early development displayed by this stem cell-based in vitro model mimics features of embryonic day 3 to day 9/10 of natural development. Thus, this platform represents a tractable model system to contribute to the basic understanding of cellular and molecular mechanisms governing early embryonic events in humans and to provide valuable insights into the design of differentiation protocols for human stem cells in clinical applications.

developmental biology↗

bioRxiv2021

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by inhibiting senescence and mesenchymal-epithelial transition, processes involved in endometrial breakdown and regeneration, respectively. Accelerated decidualization resulted in entrapment of co-cultured human blastocysts in a largely static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings demonstrate that senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

developmental biology↗

bioRxiv2020

Mechanism of cell polarisation and first lineage segregation in the human embryo

The formation of differential cell lineages in the mammalian blastocyst from the totipotent zygote is crucial for implantation and the success of the whole pregnancy. The first lineage segregation generates the polarised trophectoderm (TE) tissue, which forms the placenta, and the apolar inner cell mass (ICM), which mainly gives rise to all foetal tissues and also the yolk sac1-3. The mechanism underlying this cell fate segregation has been extensively studied in the mouse embryo4,5. However, when and how it takes place in the human embryo remains unclear. Here, using time-lapse imaging and 325 surplus human embryos, we provide a detailed characterisation of morphological events and transcription factor expression and localisation to understand how they lead to the first lineage segregation in human embryogenesis. We show that the first lineage segregation of the human embryo is triggered by cell polarisation that occurs at the 8-cell stage in two sequential steps. In the first step, F-actin becomes apically polarised concomitantly with embryo compaction. In the second step, the Par complex becomes polarised to form the apical cellular domain. Mechanistically, we show that activation of Phospholipase C (PLC) triggers actin polarisation and is therefore essential for apical domain formation, as is the case in mouse embryos6. Finally, we show that, in contrast to the mouse embryo, the key extra-embryonic determinant GATA37,8 is expressed not only in extra-embryonic lineage precursors upon blastocyst formation. However, the cell polarity machinery enhances the expression and nuclear accumulation of GATA3. In summary, our results demonstrate for the first time that cell polarisation reinforces the first lineage segregation in the human embryo.

developmental biology↗

bioRxiv2020

Transcriptional control of apical protein clustering drives de novo cell polarity establishment in the early mouse embryo

The establishment of cell polarity de novo in the early mammalian embryo triggers the transition from totipotency to differentiation to generate embryonic and extra-embryonic lineages. However, the molecular mechanisms governing the timing of cell polarity establishment remain unknown. Here, we identify stage-dependent transcription of Tfap2c and Tead4 as well as Rho GTPase signaling as key for the onset of cell polarization. Importantly, advancing their activity can induce precocious cell polarization and ectopic lineage differentiation in a cell-autonomous manner. Moreover, we show that the asymmetric clustering of apical proteins, regulated by Tfap2c-Tead4, and not actomyosin flow, mediates apical protein polarization. These findings identify the long-sought mechanism for the onset of polarization and the first lineage segregation in the mouse embryo.

developmental biology↗