Biology subjects

Hadjantonakis, A.-K.

Publications and source records attributed to Hadjantonakis, A.-K..

At least 19 recordsLinked to original sources

bioRxiv2025

L1CAM signaling through planar cell polarity generates SOX2+ metastatic progenitors in lung adenocarcinoma

Plasticity transitions during carcinoma progression generate fetal-like progenitor states with metastatic capacity. How these progenitors emerge and persist during tumor progression remains unknown. Here, we elucidate a process that drives the emergence of SOX2+ metastatic progenitors in lung adenocarcinomas (LUAD). LUAD cells in the tumor invasive front and distant metastases express the cell adhesion molecule L1CAM, a marker of regenerative epithelial progenitors and a mediator of cell-basement membrane and cell-cell interactions. L1CAM-mediated adhesion to perivascular basement membrane is known to stimulate the proliferation of extravasated micrometastatic cells. We now identify a distinct and broader role of L1CAM as promoter of the SOX2+ LUAD progenitor state. We show that L1CAM at cell-cell interfaces promotes the assembly of the planar cell polarity (PCP) complex in metastatic LUAD progenitors. L1CAM-dependent PCP acting through a non-canonical WNT signaling activates c-Jun, which cooperates with the chromatin remodeling factor CHD1 to drive SOX2 expression and metastatic activity. This axis sustains the tumor-initiating and regenerative capacity of LUAD progenitor cells. By illuminating the role of L1CAM and PCP signaling in the generation of SOX2+ LUAD progenitors, our findings identify potential new targets to treat metastatic cancer.

cancer biology↗

bioRxiv2025

Transcription factors SP5 and SP8 drive primary cilia formation

While specific transcription factors are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multi-omics comparison of the transcriptomes of ciliated and non-ciliated embryonic cells to identify transcription factors upregulated in ciliated cells, and conditional genetics in mouse embryos and stem cells to demonstrate that SP5/8 regulate cilia formation and gene expression. In Sp5/8 mutant embryos primary and motile cilia are shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 is sufficient to induce primary cilia in unciliated cells. This work opens new avenues for studying cilia assembly using stem cell models and offers new insights into human ciliopathies.

developmental biology↗

bioRxiv2024

Donor embryonic stem cells impede host epiblast specification in 8-cell stage chimeras by crowding and FGF4 signalling

During mouse embryo compaction, outer cells become trophectoderm, while inner cells form the inner cell mass (ICM), later differentiating into primitive endoderm and epiblast - source of embryonic stem cells (ESCs) - during blastocyst formation. Trophectoderm specification is driven by position-governed polarisation, while primitive endoderm specification is positively regulated by FGF4 signalling from the ICM and epiblast. When injected into an 8-cell stage morula, ESCs can exclude host cells from the epiblast, leading to mice derived entirely from these cells. While evidence suggests roles for ESC-produced FGF4 and physical crowding in host cell displacement from the ICM, the interplay between these possible mechanisms has yet to be dissected, in part due to the lack of studies using Fgf4-/- ESCs. Here, we combine chimaera titration assays with mathematical modelling to study these mechanisms of host cell displacement. Both Fgf4+/+ and Fgf4-/- ESCs displaced host cells from the epiblast, while only Fgf4-/- injected embryos reduced primitive endoderm and increased trophectoderm, indicating sequential exclusion by displacement crowding followed by FGF4 signalling. SUMMARY STATEMENTIn mouse blastocyst chimeras, donor cells displace host cells from the epiblast by a combination of crowding and FGF4 signalling.

developmental biology↗

bioRxiv2024

Axin1 and Axin2 regulate the WNT-signaling landscape to promote distinct mesoderm programs

How distinct mesodermal lineages - extraembryonic, lateral, intermediate, paraxial and axial - are specified from pluripotent epiblast during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/{beta}-catenin pathway, we have uncovered new roles for WNT signaling in the determination of mesodermal fates. We undertook complementary approaches to dissect the role of WNT signaling that augmented a detailed analysis of Axin1;Axin2 mutant mouse embryos, including single-cell and single-embryo transcriptomics, with in vitro pluripotent Epiblast-Like Cell differentiation assays. This strategy allowed us to reveal two layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors, and thereafter, WNT amplifies and cooperates with BMP/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior streak derivatives or anterior streak derivatives, respectively. We propose that Axin1 and Axin2 prevent aberrant differentiation of pluripotent epiblast cells into mesoderm by spatially and temporally regulating WNT signaling levels.

developmental biology↗

bioRxiv2024

Identification of a mesodermal progenitor for the pro-definitive angio-hematopoietic lineage

Mammalian hematopoietic cells arise from mesodermal progenitors in a close developmental relationship with endothelium, and along three distinct cell lineages known as primitive, pro-definitive, and definitive hematopoiesis. However, the developmental hierarchy between mesodermal progenitors, endothelium, and blood cell lineages is incompletely understood. We report here the identification in mouse gastrula and human (h)iPSC cultures of a population of CXCR4+ primitive streak stage mesodermal progenitors that give rise to yolk sac endothelium, blood islands, yolk sac hematopoiesis, and resident macrophages, corresponding to the pro-definitive lineage. Strikingy, this progenitor does not give rise to primitive erythropoiesis or to caudal endothelium and definitive hematopoiesis. Interestingly however, the pro-definitive progenitor population also gives rise to rostral endothelium that persists in adults. Finally, pro-definitive progenitor-derived endothelium is the first and main source of macrophages in embryo and hiPSC cultures, both directly and via previously described multipotent Erythro-Myeloid Progenitors. The identification and isolation of this mesodermal progenitor defines a revised pro-definitive angio-hematopoietic lineage and provides a framework for resident macrophage and endothelial differentiation relevant to disease pathophysiology and their manipulation for therapeutic purposes. One sentence summaryIdentification in gastrulating embryo and hiPSC cultures of a mesodermal progenitor for yolk sac and rostral endothelial cells, early macrophages, and Erythro-Myeloid Progenitors characterize pro-definitive hematopoiesis and provide a revised framework for early angio-hematopoietic development. Graphical abstractSchematic represents the progeny of angio-hematopoietic progenitors at the posterior primitive streak. Developmental timing of CXCR4 expression distinguishes i) CXCR4- primitive hematopoiesis, ii) Early CXCR4+ angio-hematopoietic progenitors (EXAP) (purple) which express CXCR4 between E6.5 and E7.5 and give rise to rostral vessels and resident macrophages through YS hemogenic endothelium, and iii) late CXCR4+ intraembryonic angio-hematopoietic progenitors (blue) which acquire expression of CXCR4 around E8 and give rise to definitive hematopoiesis through an intraembryonic hemogenic endothelium. Abbreviations: E (Mouse Embryonic Day); YS (Yolk Sac); Hemog. (Hemogenic); Endo. (Endothelium); Defi. (Definitive). O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/609533v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@afb5fdorg.highwire.dtl.DTLVardef@1d4f772org.highwire.dtl.DTLVardef@c173d9org.highwire.dtl.DTLVardef@9cc17d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

ETV4 and ETV5 Orchestrate FGF-Mediated Lineage Specification and Epiblast Maturation during Early Mouse Development

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signaling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are critical mediators of FGF signaling in cell lineage specification and maturation in mouse embryos. We show that loss of Etv5 compromises primitive endoderm formation at pre-implantation stages. Furthermore, Etv4/5 deficiency delays naive pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, Etv4/5 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 signaling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development. Summary statementFGF signaling effectors ETV4/5 regulate lineage specification and embryo patterning in mice, affecting primitive endoderm formation and pluripotency exit.

developmental biology↗

bioRxiv2023

Tgfbr1 regulates lateral plate mesoderm and endoderm reorganization during the trunk to tail transition

During the trunk to tail transition the mammalian embryo builds the outlets for the intestinal and urogenital tracts, lays down the primordia for the hindlimb and external genitalia, and switches from the epiblast/primitive streak to the tailbud as the driver of axial extension. Genetic and molecular data indicate that Tgfbr1 is a key regulator of the trunk to tail transition. Tgfbr1 has been shown to control the switch of the neuro mesodermal-competent cells from the epiblast to the chordo-neural hinge to generate the tail bud. We now show that Tgfbr1 signaling also controls the remodeling of the lateral plate mesoderm (LPM) and of the embryonic endoderm associated with the trunk to tail transition. In the absence of Tgfbr1 the two LPM layers do not converge at the end of the trunk, extending instead as separate layers enclosing the celomic cavity until the caudal embryonic extremity, and failing to activate markers of primordia for the hindlimb and external genitalia. However, this extended LPM, does not exhibit the molecular signatures characteristic of this tissue in the trunk. The vascular remodeling involving the dorsal aorta and the umbilical artery leading to the connection between embryonic and extraembryonic circulation was also affected in the Tgfbr1 mutant embryos. Similar alterations in the LPM and vascular system were also observed in Isl1 null mutants, indicating that this factor acts in the regulatory cascade downstream of Tgfbr1 in LPM-derived tissues. In addition, in the absence of Tgfbr1 the embryonic endoderm fails to expand to form the endodermal cloaca and to extend posteriorly to generate the tail gut. We present evidence suggesting that the remodeling activity of Tgfbr1 in the LPM and endoderm results from the control of the posterior primitive streak fate after its regression during the trunk to tail transition. Our data, together with previously reported observations, place Tgfbr1 at the top of the regulatory processes controlling the trunk to tail transition.

developmental biology↗

bioRxiv2023

Systematic mapping and modeling of 3D enhancer-promoter interactions in early mouse embryonic lineages reveal regulatory principles that determine the levels and cell-type specificity of gene expression

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or "hubness" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors. HIGHLIGHTS- Cell lines representing early embryonic lineages undergo drastic enhancer remodeling and fine-scale 3D chromatin reorganization - Highly interacting 3D hubs strongly enrich for highly expressed, cell-type specific and essential genes - 3D chromatin features greatly improve prediction of cell-type specific gene expression compared to 1D promoter features - In silico and experimental perturbations identify novel enhancers regulating the expression of two or more genes in early embryonic lineages

molecular biology↗

bioRxiv2023

The covariance environment defines cellular niches for spatial inference

The tsunami of new multiplexed spatial profiling technologies has opened a range of computational challenges focused on leveraging these powerful data for biological discovery. A key challenge underlying computation is a suitable representation for features of cellular niches. Here, we develop the covariance environment (COVET), a representation that can capture the rich, continuous multivariate nature of cellular niches by capturing the gene-gene covariate structure across cells in the niche, which can reflect the cell-cell communication between them. We define a principled optimal transport-based distance metric between COVET niches and develop a computationally efficient approximation to this metric that can scale to millions of cells. Using COVET to encode spatial context, we develop environmental variational inference (ENVI), a conditional variational autoencoder that jointly embeds spatial and single-cell RNA-seq data into a latent space. Two distinct decoders either impute gene expression across spatial modality, or project spatial information onto dissociated single-cell data. We show that ENVI is not only superior in the imputation of gene expression but is also able to infer spatial context to disassociated single-cell genomics data.

bioinformatics↗

bioRxiv2023

Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities

Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and eXtraembryonic ENdoderm (XEN) stem cells - in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. The dominant PrE transcriptional program, safeguarded by Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM.

developmental biology↗

bioRxiv2023

ZFP281 coordinates DNMT3 and TET1 for transcriptional and epigenetic control in pluripotent state transitions

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions. In BriefThe naive, formative, and primed pluripotent states and their interconversions recapitulate pluripotency continuum during early development. Huang and colleagues investigated the transcriptional programs during successive pluripotent state transitions and revealed an essential role for ZFP281 in coordinating DNMT3A/3B and TET1 to establish the DNA methylation and gene expression programs during the transitions. HighlightsO_LIZFP281 activates Dnmt3a/3b in vitro in pluripotent stem cells and in vivo in epiblast. C_LIO_LIZFP281 and TET1 undergo bimodal chromatin occupancy in pluripotent state transitions. C_LIO_LIChromatin-binding of ZFP281 and TET1 depends on the formation of R-loops at promoters. C_LIO_LIZFP281 is necessary for the establishment and maintenance of primed pluripotency. C_LI

developmental biology↗

bioRxiv2023

Evolution of promoter-proximal pausing enabled a new layer of transcription control

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. Unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites that transitioned to a longer-lived, focused pause in metazoans. This event coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

evolutionary biology↗

bioRxiv2023

Distinct tumor architectures for metastatic colonization of the brain

Brain metastasis is a dismal cancer complication, hinging on the initial survival and outgrowth of disseminated cancer cells. To understand these crucial early stages of colonization, we investigated two prevalent sources of cerebral relapse, triple-negative (TNBC) and HER2+ breast cancer (HER2BC). We show that these tumor types colonize the brain aggressively, yet with distinct tumor architectures, stromal interfaces, and autocrine growth programs. TNBC forms perivascular sheaths with diffusive contact with astrocytes and microglia. In contrast, HER2BC forms compact spheroids prompted by autonomous extracellular matrix components and segregating stromal cells to their periphery. Single-cell transcriptomic dissection reveals canonical Alzheimers disease-associated microglia (DAM) responses. Differential engagement of tumor-DAM signaling through the receptor AXL suggests specific pro-metastatic functions of the tumor architecture in both TNBC perivascular and HER2BC spheroidal colonies. The distinct spatial features of these two highly efficient modes of brain colonization have relevance for leveraging the stroma to treat brain metastasis.

cancer biology↗

bioRxiv2022

Amino acid intake strategies define pluripotent cell states

Mammalian pre-implantation development is associated with striking metabolic robustness, as embryos can develop in a wide variety of nutrient conditions including even the complete absence of soluble amino acids. Here, we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of pre-implantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis alongside a robust lysosomal digestive system. Upon transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant upon exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the pre-implantation epiblast. More broadly, we find that all lineages of the pre-implantation blastocysts exhibit constitutive macropinocytotic protein uptake and digestion. Together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of pre-implantation development and provide insight into the catabolic strategies that enable embryos to sustain viability prior to implantation.

developmental biology↗

bioRxiv2022

3D genome topologies distinguish pluripotent epiblast and primitive endoderm cells in the mouse blastocyst

The development of embryonic cell lineages is tightly controlled by transcription factors that regulate gene expression and chromatin organisation. To investigate the specialisation of 3D genome structure in pluripotent or extra-embryonic endoderm lineages, we applied Genome Architecture Mapping (GAM) in embryonic stem (ES) cells, extra-embryonic endoderm (XEN) stem cells, and in their in vivo counterparts, the epiblast (Epi) and primitive endoderm (PrE) cells, respectively. We discover extensive differences in 3D genome topology including the formation domain boundaries that differ between Epi and PrE lineages, both in vivo and in vitro, at lineage commitment genes. In ES cells, Sox2 contacts other active regions enriched for NANOG and SOX2 binding sites. PrE-specific genes, such as Lama1 and Gata6, form repressive chromatin hubs in ES cells. Lama1 activation in XEN or PrE cells coincides with its extensive decondensation. Putative binding sites for OCT4 and SNAIL, or GATA4/6, distinguish chromatin contacts unique to embryonic or extra-embryonic lineages, respectively. Overall, 3D genome folding is highly specialised in early development, especially at genes encoding factors driving lineage identity. HighlightsO_LIES and XEN cells have specialised 3D genome structures C_LIO_LIGAM applied in the blastocyst distinguishes Epi and PrE genome structures C_LIO_LILineage specific genes establish cell-type specific chromatin contacts C_LIO_LISpecific chromatin contacts feature putative bindings sites for GATA4/6 in XEN cells and SNAIL in ES cells C_LI

developmental biology↗

bioRxiv2022

RNA polymerase II and PARP1 shape enhancer-promoter contacts

How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we studied enhancer-promoter contact architecture and communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription, and perturbations to transcription-associated proteins and thousands of candidate enhancers. Contact frequency between functionally validated enhancer-promoter pairs was most enriched near the +1 and +2 nucleosomes at enhancers and target promoters, indicating that functional enhancer-promoter pairs spend time in close physical proximity. Blocking RNA polymerase II (Pol II) caused major disruptions to enhancer-promoter contacts. Paused Pol II occupancy and the enzymatic activity of poly (ADP-ribose) polymerase 1 (PARP1) stabilized enhancer-promoter contacts. Based on our findings, we propose an updated model that couples transcriptional dynamics and enhancer-promoter communication.

genomics↗

bioRxiv2022

A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT

Epithelial-to-Mesenchymal Transition (EMT) is a fundamental process in which epithelial cells acquire mesenchymal phenotypes and the ability to migrate. EMT is the hallmark of gastrulation, an evolutionarily conserved developmental process. In mammals, epiblast cells lose pluripotency and ingress at the primitive streak to form mesoderm. Cell exit from the epiblast epithelial layer and the associated EMT are dynamically regulated processes involving a stereotypical sequence of cell behaviors. 3D time-lapse imaging of gastrulating mouse embryos combined with cell and tissue scale data analyses revealed the stochastic-like ingression of epiblast cells at the primitive streak. Ingressing cells constrict their apical surfaces in a pulsed ratchet-like fashion through asynchronous shrinkage of apical junctions. A quantitative analyses of the distribution of apical proteins, revealed the anisotropic and complementary distribution of members of the actomyosin network and Crumbs2 complexes, potential regulators of asynchronous shrinkage of cell junctions. The analysis of mutants demonstrated a requirement for Crumbs2 in Myosin2 localization and activity at apical junctions, and as a candidate for regulating actomyosin anisotropy.

developmental biology↗

bioRxiv2022

RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription

Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been studied predominantly in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we find that loss of RNA Pol II pausing stalls pluripotent state transitions in the epiblast of the early mouse embryo. Using Nelfb-/- mice and a novel NELFB- degron mouse embryonic stem cells, we show that mouse ES cells (mESCs) representing the naive state of pluripotency successfully initiate a transition program, but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. Consistently, we find an increase in chromatin-associated NELF during pluripotency transitions. Overall, our work reveals the molecular and phenotypic roles of Pol II pausing in pluripotency and introduces Pol II pausing as a modulator of cell state transitions.

molecular biology↗