Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination.

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Red2Flpe-SCON: a versatile, multicolor strategy for generating mosaic conditional knockout mice.

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.

FOXA2 rewires AP-1 for transcriptional reprogramming and lineage plasticity in prostate cancer.

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity.

Plasticity and lineage commitment of individual TH1 cells are determined by stable T-bet expression quantities.

T helper 1 (TH1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated TH1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus-induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the TH2 lineage: T-bet quantities were inversely correlated with the ability to express the TH2 lineage-specifying transcription factor GATA-3 and TH2 cytokines. Reprogramed TH1 cells acquired graded mixed TH1 + TH2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated TH1 cells was essential to ensure TH1 cell stability. Thus, innate cytokine signals regulate TH1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

Cardiac Progenitor Cells of the First and Second Heart Fields.

The heart forms from the first and second heart fields, which contribute to distinct regions of the myocardium. This is supported by clonal analyses, which identify corresponding first and second cardiac cell lineages in the heart. Progenitor cells of the second heart field and its sub-domains are controlled by a gene regulatory network and signaling pathways, which determine their behavior. Multipotent cells in this field can also contribute cardiac endothelial and smooth muscle cells. Furthermore, the skeletal muscles of the head and neck are clonally related to myocardial cells that form the arterial and venous poles of the heart. These lineage relationships, together with the genes that regulate the heart fields, have major implications for congenital heart disease.

The matrisome landscape controlling in vivo germ cell fates.

The developmental fate of cells is regulated by intrinsic factors and the extracellular environment. The extracellular matrix (matrisome) delivers chemical and mechanical cues that can modify cellular development. However, comprehensive understanding of how matrisome factors control cells in vivo is lacking. Here we show that specific matrisome factors act individually and collectively to control germ cell development. Surveying development of undifferentiated germline stem cells through to mature oocytes in the Caenorhabditis elegans germ line enabled holistic functional analysis of 443 conserved matrisome-coding genes. Using high-content imaging, 3D reconstruction, and cell behavior analysis, we identify 321 matrisome genes that impact germ cell development, the majority of which (>80%) are undescribed. Our analysis identifies key matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior. Further, our results demonstrate that germ cell development requires continual remodeling of the matrisome landscape. Together, this study provides a comprehensive platform for deciphering how extracellular signaling controls cellular development and anticipate this will establish new opportunities for manipulating cell fates.

Investigating the basis of lineage decisions and developmental trajectories in the dorsal spinal cord through pseudotime analyses.

Dorsal interneurons (dIs) in the spinal cord encode the perception of touch, pain, heat, itchiness and proprioception. Previous studies using genetic strategies in animal models have revealed important insights into dI development, but the molecular details of how dIs arise as distinct populations of neurons remain incomplete. We have developed a resource to investigate dI fate specification by combining a single-cell RNA-Seq atlas of mouse embryonic stem cell-derived dIs with pseudotime analyses. To validate this in silico resource as a useful tool, we used it to first identify genes that are candidates for directing the transition states that lead to distinct dI lineage trajectories, and then validated them using in situ hybridization analyses in the developing mouse spinal cord in vivo. We have also identified an endpoint of the dI5 lineage trajectory and found that dIs become more transcriptionally homogeneous during terminal differentiation. This study introduces a valuable tool for further discovery about the timing of gene expression during dI differentiation and demonstrates its utility in clarifying dI lineage relationships.

Branching topology of the human embryo transcriptome revealed by Entropy Sort Feature Weighting.

Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after subsetting to highly variable genes (HVGs). Here, we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous Entropy Sort Feature Weighting (cESFW) outperforms HVG selection in distinguishing cell-state-specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from eight-cell to post-implantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development, while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression, we provide markers for staging progression from morula to blastocyst.

Comprehensive data mining reveals RTK/RAS signaling pathway as a promoter of prostate cancer lineage plasticity through transcription factors and CNV.

Prostate cancer lineage plasticity is a key driver in the transition to neuroendocrine prostate cancer (NEPC), and the RTK/RAS signaling pathway is a well-established cancer pathway. Nevertheless, the comprehensive link between the RTK/RAS signaling pathway and lineage plasticity has received limited investigation. In particular, the intricate regulatory network governing the interplay between RTK/RAS and lineage plasticity remains largely unexplored. The multi-omics data were clustered with the coefficient of argument and neighbor joining algorithm. Subsequently, the clustered results were analyzed utilizing the GSEA, gene sets related to stemness, multi-lineage state datasets, and canonical cancer pathway gene sets. Finally, a comprehensive exploration of the data based on the ssGSEA, WGCNA, GSEA, VIPER, prostate cancer scRNA-seq data, and the GPSAdb database was conducted. Among the six modules in the clustering results, there are 300 overlapping genes, including 3 previously unreported prostate cancer genes that were validated to be upregulated in prostate cancer through RT-qPCR. Function Module 6 shows a positive correlation with prostate cancer cell stemness, multi-lineage states, and the RTK/RAS signaling pathway. Additionally, the 19 leading-edge genes of the RTK/RAS signaling pathway promote prostate cancer lineage plasticity through a complex network of transcriptional regulation and copy number variations. In the transcriptional regulation network, TP63 and FOXO1 act as suppressors of prostate cancer lineage plasticity, whereas RORC exerts a promoting effect. This study provides a comprehensive perspective on the role of the RTK/RAS pathway in prostate cancer lineage plasticity and offers new clues for the treatment of NEPC.

A new dawn for the study of cell type evolution.

Animal evolution is influenced by the emergence of new cell types, yet our understanding of this process remains elusive. This prompts the need for a broader exploration across diverse research organisms, facilitated by recent breakthroughs, such as gene editing tools and single-cell genomics. Essential to our understanding of cell type evolution is the accurate identification of homologous cells. We delve into the significance of considering developmental ontogeny and potential pitfalls when drawing conclusions about cell type homology. Additionally, we highlight recent discoveries in the study of cell type evolution through the application of single-cell transcriptomics and pinpoint areas ripe for further exploration.

Scuphr: A probabilistic framework for cell lineage tree reconstruction.

Cell lineage tree reconstruction methods are developed for various tasks, such as investigating the development, differentiation, and cancer progression. Single-cell sequencing technologies enable more thorough analysis with higher resolution. We present Scuphr, a distance-based cell lineage tree reconstruction method using bulk and single-cell DNA sequencing data from healthy tissues. Common challenges of single-cell DNA sequencing, such as allelic dropouts and amplification errors, are included in Scuphr. Scuphr computes the distance between cell pairs and reconstructs the lineage tree using the neighbor-joining algorithm. With its embarrassingly parallel design, Scuphr can do faster analysis than the state-of-the-art methods while obtaining better accuracy. The method's robustness is investigated using various synthetic datasets and a biological dataset of 18 cells.

Integrating phylogenies into single-cell RNA sequencing analysis allows comparisons across species, genes, and cells.

Comparisons of single-cell RNA sequencing (scRNA-seq) data across species can reveal links between cellular gene expression and the evolution of cell functions, features, and phenotypes. These comparisons evoke evolutionary histories, as depicted by phylogenetic trees, that define relationships between species, genes, and cells. This Essay considers each of these in turn, laying out challenges and solutions derived from a phylogenetic comparative approach and relating these solutions to previously proposed methods for the pairwise alignment of cellular dimensional maps. This Essay contends that species trees, gene trees, cell phylogenies, and cell lineages can all be reconciled as descriptions of the same concept-the tree of cellular life. By integrating phylogenetic approaches into scRNA-seq analyses, challenges for building informed comparisons across species can be overcome, and hypotheses about gene and cell evolution can be robustly tested.

Emx2 lineage tracing reveals antecedent patterns of planar polarity in the mouse inner ear.

The planar polarized organization of hair cells in the vestibular maculae is unique because these sensory organs contain two groups of cells with oppositely oriented stereociliary bundles that meet at a line of polarity reversal (LPR). EMX2 is a transcription factor expressed by one hair cell group that reverses the orientation of their bundles, thereby forming the LPR. We generated Emx2-CreERt2 transgenic mice for genetic lineage tracing and demonstrate Emx2 expression before hair cell specification when the nascent utricle and saccule constitute a continuous prosensory domain. Precursors labeled by Emx2-CreERt2 at this stage give rise to hair cells located along one side of the LPR in the mature utricle or saccule, indicating that this boundary is first established in the prosensory domain. Consistent with this, Emx2-CreERt2 lineage tracing in Dreher mutants, where the utricle and saccule fail to segregate, labels a continuous field of cells along one side of a fused utriculo-saccular-cochlear organ. These observations reveal that LPR positioning is pre-determined in the developing prosensory domain, and that EMX2 expression defines lineages of hair cells with oppositely oriented stereociliary bundles.

The single-cell chromatin landscape in gonadal cell lineage specification.

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Influence of Human Bone Marrow Mesenchymal Stem Cells Secretome from Acute Myeloid Leukemia Patients on the Proliferation and Death of K562 and K562-Lucena Leukemia Cell Lineages.

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.

MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells.

Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Enhancer switching in cell lineage priming is linked to eRNA, Brg1's AT-hook, and SWI/SNF recruitment.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.

Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells1. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the 'sign' of the effect is wholly dependent on the environment.