Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency.

The gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.This research was funded by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, European Commission (contract no. 200720, EuroSyStem) and the Louis Jeantet Foundation. The Cambridge Stem Cell Institute receives core support from the Wellcome Trust and the Medical Research Council. AS is a Medical Research Council Professor

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.CRUK, Wellcome Trust, MRC, BBSRC, EMBL, E

Multi-omics characterisation of mouse gastrulation and organogenesis at single-cell resolution

Soon after implantation, the mammalian embryo needs to instruct the specification of the epiblast precursors required for the formation of the foetal tissues. At the exit from pluripotency, global epigenetic and transcriptional remodelling occurs. These changes are essential for gastrulation, the process by which all three germ layers - ectoderm, mesoderm, and endoderm - are specified. Recent advances in single-cell sequencing technologies have allowed the characterisation of the transcriptional and epigenetic changes during mouse gastrulation and early organogenesis. However, the precise molecular mechanisms that control cell fate decisions are still poorly understood. Therefore, my PhD project aimed to understand further how genetically identical cells can be primed for differentiation and specification. My PhD work's main focus was to characterise (1) the role of the spatial environment and (2) DNA methylation in cell lineage priming and specification during embryonic development. To better understand the spatial environment's role on cell fate specification (Aim 1), we used the image-based single-cell transcriptomics method, seqFISH, to precisely measure the mRNA abundance of 387 carefully selected target genes in mouse embryos. Therefore, I developed a new cell segmentation strategy and performed seqFISH on E8.5 mouse embryo tissue sections to characterise the roles of the spatial environment on cell fate specification during organogenesis. Joint analysis of the seqFISH data with previously published scRNA-seq data allowed us to address biological questions related to spatial coherence of cell types, the emergence of the midbrain-hindbrain boundary and gut tube organogenesis. Strikingly, we demonstrated that the spatial patterning of the gut tube is associated with distinct organ primordia. The spatial atlas uncovers axes of resolution that cannot be reconstructed from single-cell RNA sequencing data, for example, we observe the dorsal-ventral separation of oesophageal and tracheal progenitor populations 24-hours earlier than previously appreciated. The manipulation of DNA methylation levels by knockout of methylation modifiers in vivo results in embryonic lethality shortly after gastrulation, highlighting the essential role of fine-tuned DNA methylation in mammalian development. However, how DNA methylation influences cell fate decisions is still poorly understood. To address this, I used deletion of DNA methylation enzymes in mouse embryos and stem cell culture systems to ask whether DNA methylation is required for cell fate choices (Aim 2). I showed that DNA methylation is necessary to exit pluripotency and restrict the differentiation potential to extraembryonic lineages. Notably, we showed that the loss of TET dioxygenases results in mesodermal differentiation defects, which was particularly pronounced for mature blood cells and cardiomyocytes. Strikingly, we demonstrated that TETs are required to demethylate and activate mesoderm specific enhancers during cellular differentiation, using single-cell nucleosome, methylome and transcriptome sequencing (scNMT-seq). Hence, we hypothesised that the inability of the cells to reprogramme the epigenome causes the differentiation defect detected. The results demonstrate the essential role of active DNA demethylation for lineage specification. We are currently expanding on these intriguing observations by identifying lineage-specific DNA methylation defects in the blood lineage of Tet1/2/3 TKO chimeras. The results will reveal core functions of DNA demethylation during cell lineage specification and establishment of developmental competence. In sum, my PhD project provides a detailed study of the role of the spatial environment on transcriptional regulation (Aim 1) and the causal relationship between DNA methylation and cell fate in early mammalian embryogenesis (Aim 2)

Dynamic DNA methylation turnover at the exit of pluripotency epigenetically primes gene regulatory elements for hematopoietic lineage specification

Epigenetic mechanisms govern developmental cell fate decisions, but how DNA methylation coordinates with chromatin structure and three-dimensional DNA folding to enact cell-type specific gene expression programmes remains poorly understood. Here, we use mouse embryonic stem and epiblast-like cells deficient for 5-methyl cytosine or its oxidative derivatives (5-hydroxy-, 5-formyl- and 5-carboxy-cytosine) to dissect the gene regulatory mechanisms that control cell lineage specification at the exit of pluripotency. Genetic ablation of either DNA methyltransferase ( Dnmt ) or Ten-eleven-translocation ( Tet ) activity yielded largely distinct sets of dysregulated genes, revealing divergent transcriptional defects upon perturbation of individual branches of the DNA cytosine methylation cycle. Unexpectedly, we found that disrupting DNA methylation or oxidation interferes with key enhancer features, including chromatin accessibility, enhancer-characteristic histone modifications, and long-range chromatin interactions with putative target genes. In addition to affecting transcription of select genes in pluripotent stem cells, we observe impaired enhancer priming, including a loss of three-dimensional interactions, at regulatory elements associated with key lineage-specifying genes that are required later in development, as we demonstrate for the key hematopoietic genes Klf1 and Lyl1 . Consistently, we observe impaired transcriptional activation of blood genes during embryoid body differentiation of knockout cells. Our findings identify a novel role for the dynamic turnover of DNA methylation at the exit of pluripotency to establish and maintain chromatin states that epigenetically prime enhancers for later activation during developmental cell diversification. Highlights We perform a detailed epigenetic characterisation of the mouse embryonic stem cell (ESC) to epiblast-like cell (EpiLC) transition in wild type, Tet triple-knockout (TKO) and Dnmt TKO lines and develop a novel clustering approach to interrogate the data. Tet TKO reduces H3K4me1 and H3K27ac levels across enhancer elements upon pluripotency exit whilst Dnmt TKO affects only H3K4me1 levels, suggesting a novel role for oxidative derivatives in H3K4me1 deposition. Tet TKO and Dnmt TKO affect enhancer priming in EpiLCs which is associated with failure to upregulate hematopoietic genes upon differentiation. Long-range chromosomal interactions between primed enhancers and their target genes are weakened in both Dnmt and Tet TKO

Investing in vision:Innovation in retinal therapeutics and the influence on venture capital investment

Since the groundbreaking approval of the first anti-VEGF therapy in 2004, the retinal therapeutics field has undergone a remarkable transformation, witnessing a surge in novel, disease-modifying therapeutics for a broad spectrum of retinal diseases, extending beyond exudative VEGF-driven conditions. The surge in scientific advancement and the pressing, unmet, medical need have captured the attention of venture capital investors, who have collectively invested close to $10 billion in research and development of new retinal therapeutics between 2004 and 2023. Notably, the field of exudative diseases has gradually shifted away from trying to outcompete anti-VEGF therapeutics towards lowering the overall treatment burden by reducing injection frequency. Simultaneously, a new era has emerged in the non-exudative field, targeting prevalent conditions like dry AMD and rare indications such as Retinitis pigmentosa. This has led to promising drug candidates in development, culminating in the landmark approval of Luxturna for a rare form of Retinitis pigmentosa. The validation of new mechanisms, such as the complement pathway in dry AMD has paved the way for the approvals of Syvovre (Apellis) and Izervay (Iveric/Astellas), marking the first two therapies for this condition. In this comprehensive review, we share our view on the cumulative lessons from the past two decades in developing retinal therapeutics, covering both positive achievements and challenges. We also contextualize the investments, strategic partnering deals, and acquisitions of biotech companies, pharmaceutical companies venture capital investors in retinal therapeutics, respectively. Finally, we provide an outlook and potentially a forward-looking roadmap on novel retinal therapeutics, highlighting the emergence of potential new intervention strategies, such as cell-based therapies, gene editing, and combination therapies. We conclude that upcoming developments have the potential to further stimulate venture capital investments, which ultimately could facilitate the development and delivery of new therapies to patients in need.</p