ABSTRACT:JAK2V617F is one of the most common mutations in clonal hematopoiesis of indeterminate potential (CHIP) and a major driver of myeloproliferative neoplasms (MPNs). To determine the impact of a low-frequency JAK2V617F clone on both the hematopoietic system and the bone marrow (BM) stroma, we developed a traceable murine MPN model, in which whole BM transplantation (BMT) was performed using CD45.2 5.0 × 106 JAK2V617F donor cells transplanted into unconditioned CD45.1 recipient mice. BMT recipients developed a polycythemia vera-like phenotype (elevated hematocrit and leukocytosis) with a 2.7% average donor cell chimerism in peripheral blood. Eight months after BMT, RNA sequencing (RNA-seq) analysis of BM cells sorted according to CD45.1/CD45.2 expression showed significant upregulation of early erythroblast- and myeloid cell-specific transcripts, and downregulation of lymphoid transcripts in donor-derived cells compared to controls. Surprisingly, recipient-derived cells also showed upregulation of myeloid- and erythroblast-related transcripts, indicating a skewing of the non-JAK2V617F-carrying recipient hematopoietic system toward an MPN-like phenotype. In addition, RNA-seq analysis of the BM stroma from JAK2V617F BMT recipients indicated significant loss of osteomesenchymal transcripts. Consistently, micro-computed tomography imaging indicated loss of trabecular bone. In sum, our results indicate that low-frequency MPN-driving cells in unconditioned recipients not only impact hematopoiesis-supporting stroma but also profoundly influence unmutated cells, uniquely altering their transcriptomic and phenotypic profiles. These observations are challenging our current understanding of the etiology and therapeutic approaches to MPNs and other CHIP-associated diseases.

Efficient drug delivery to glioblastoma (GBM) is a major obstacle as the blood-brain barrier (BBB) and the blood-tumor barrier (BTB) prevent passage of the majority of chemotherapies into the brain. Here, we identified a transcriptional 12-gene signature associated with the BTB in GBM. We identified CDH5 as a core molecule in this set and confirmed its expression in GBM vasculature using transcriptomics and immunostaining of patient specimens. The indirubin-derivative, 6-bromoindirubin acetoxime (BIA), down-regulates CDH5 and other BTB signature genes, causing endothelial barrier disruption in vitro and in murine GBM xenograft models. Treatment with BIA increased intratumoral cisplatin accumulation and potentiated DNA damage by targeting DNA repair pathways. Last, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved cisplatin efficacy in murine GBM. Our work reveals potential targets of the BTB and the bifunctional properties of BIA as a BTB modulator and a potentiator of chemotherapy, supporting its further development.

JAK2V617F is a major driver mutation in myeloproliferative neoplasms (MPN) and JAK2 inhibitors can alleviate the disease burden for patients with MPN. However, questions remain about the mechanistic role of JAK2V617F in driving the pleiotropy of phenotypes which range from low-variant allele frequency (VAF) clonal hematopoiesis (CHIP) to MPN to secondary AML; about the reason why highly specific JAK2 inhibitors fail to provide a curative potential; and about potential effects that the JAK2V617F clone might exert on both hematopoietic and non-hematopoietic components of the bone marrow. Models able to provide insights into these long-standing questions and inform optimized treatment strategies are critically needed. Here we characterize a previously described traceable murine JAK2V617F MPN model (Bonal et al, 2023), with the disease driven by a low-VAF JAK2V617F clone and induced in unconditioned bone marrow transplantation (BMT) recipients. BMT recipients displayed profound BM microenvironmental alterations, with loss of osteo/mesenchymal clones and trabecular bone. Our model also revealed MPN-mimicking cells arising from the host hematopoietic system and contributing to the MPN-like phenotype. Whole BMT was performed via a single tail vein injection of 5.0x106 donor cells into unconditioned C57BL/6 Ptprca (JAX#002014, CD45.1) recipient mice. Donor cells (JAK2V617F) were from a Poly I:C inducible MPN-like model created by crossing floxed-JAK2V617F (JAX#031658) with Mx1-Cre (JAX#003556) mice. All mice, including non-BMT wild type controls, were age- (10 weeks) and sex-matched. BMT of JAK2V67F clone resulted in a polycythemia vera-like phenotype (elevated hematocrit and white blood cell counts) with an average donor cell chimerism in peripheral blood of 2.74% (SD = 2.07). Eight months post-BMT, we performed bulk RNA-seq on whole BM sorted according to CD45 expression (CD45.1 recipient/control or CD45.2 donor). Gene set enrichment analysis of sorted recipient and donor fractions revealed significant upregulation of early erythroblast and downregulation of lymphoid pathways (B, T, NK cells) in donor-derived cells compared to controls. Surprisingly, recipient-derived cells showed upregulation in myeloid- (monocytes, eosinophils, neutrophils) and erythroblast-related gene sets, indicating a skewing of the non-JAK2V617F carrying recipient hematopoietic system towards an MPN-like phenotype. Confirming our RNA-seq results, flow cytometry analyses of donor-derived (CD45.2) cells showed significantly higher levels of erythroid (CD71+/Ter119+), megakaryocyte (CD42d+/CD41+), myeloid (CD11b+), and granulocyte (Gr-1+/CD66a+) progenitors, with significantly lower T- (CD3+) and B-cell (B220+/CD19+) frequencies compared to controls. The most elevated cell type originating from donor cells was a megakaryocyte progenitor (CD42d+/CD41+). The erythroid progenitor output was similar between donor-derived and non-BMT wild type control populations, suggesting the small clone within recipients drove an equal output of erythroid cells compared to a healthy non-BMT animal. Intriguingly, recipient-derived (CD45.1) cells had significantly elevated monocytic (F4/80+/CD11b+) and erythroid (CD71+/Ter119+) progenitors, with nonsignificant trends towards higher myeloid and granulocyte progenitors and lower B cells, consistent with RNA-seq. We further observed an unexpected, significant increase in erythroid (CD71+/Ter119+) cells originating from the CD45.1 recipient population in the BMT mice, suggesting that the elevation in erythrocytes and the MPN phenotype partly results from the JAK2V617F clone on the CD45.1 recipient's bone marrow function. Our results suggest that the presence of the JAK2V617F clone, even in low frequencies, can profoundly impact the host hematopoietic system to contribute to the manifestation of an MPN phenotype. Our model uncovers the impact of JAK2V617F donor cells on the host BM microenvironment and hematopoietic system, driving an MPN phenotype even with low donor cell chimerism. The observation that non-JAK2V617F carrying hematopoietic cells participate in MPN progression suggests that the MPN clone not only impacts hematopoiesis-supporting stroma, but may influence non-mutated cells, and may change our understanding and therapeutic approaches to human MPN, especially in patients with low VAFs.

Background: JAK2V617F (JAK2VF) is the primary driver mutation in classical myeloproliferative neoplasms (MPN). JAK2VF is present decades prior to diagnosis (Mitchell et.al. Nature 2022), and the JAK2VF+ clone may remodel the BM microenvironment further supporting its expansion (Curto-Garcia, et. al. Haematologica 2020). Understanding this relationship, particularly during early disease development may aid identification of new therapeutic targets to stop or revert these early changes. Human BM procured before MPN diagnosis is rarely available and most murine MPN models use myeloablative conditioning (eg., irradiation), permanently altering the BM stroma (Costa & Reagan Frontiers 2019), diminishing the clinical relevance of resultant findings. We developed an innovative translatable model of MPN with sustainable low-level disease engraftment and no requirement for recipient conditioning. By preserving the normal microenvironment, our model enabled detection of early changes within the BM stroma induced by the JAK2VF+ clone. Methods: Whole BM transplantation (BMT) was performed via a single tail vein injection into unconditioned C57BL/6 Ptprc a (JAX#002014, CD45.1+) recipient mice. Donor animals consisted of either wildtype ( wt) C57BL/6 (JAX#000664) mice or our PolyI:C inducible MPN-like model created by crossing a floxed-JAK2VF (JAX#031658) with Mx1-Cre (JAX#003556) animals (JAK2VF Parental). For all BMTs, donors and recipients were age- (10wks) and sex-matched. BMT cell concentrations ranged from 1e6 to 15e6per injection, n≥10 mice, both sexes were used per group. A cohort of non-BMT age- and sex-matched recipients were maintained as naïve controls. Recipient serial submandibular blood collections were used to examine complete blood cell counts and donor chimerism. Bulk RNA-seq transcriptomic analysis was performed on digested and lineage-negative sorted whole BM from matched naïve and 5e6 BMT recipients (n=7 and 6, respectively) to delineate stromal alterations driven by JAK2VF+ MPN clone. Qualitative x-ray analysis was performed on femurs. Results: 15e6 wt donor CD45.2 BM cells can be transplanted sustainably into unconditioned CD45.1 recipients with ~3.0% donor chimerism, maintained up to 200-days post-BMT (Fig A). Follow up secondary BMT into conditioned recipients using as few as 25e4CD45.2+ cells sorted from primary BMT recipients displayed 83% average engraftment by 150-day post BMT, confirming functional wt donor CD45.2 HSC. This successful strategy was used to perform BMT using JAK2VF parental (1e6, 5e6, and 15e6) cells. Detected chimerism followed a dose response, not exceeding an average of 10% up to >225 days. Surprisingly, with the engraftment at <10%, we observed MPN-like phenotype (elevated hemoglobin and monocytosis) except for no thrombocytosis (Fig A). To identify alterations in the BM niche, stromal cells were isolated from BMT recipients and subjected to RNA-seq. Enrichment of stromal cells was validated by confirming that 23% of the top 100 highest read coverage transcripts were encoding stromal genes (i.e. Sparc, Dcn, Col2a1, and Cxcl12). Gene set enrichment analysis (GSEA) of stromal fraction showed upregulation of osteoclastic differentiation and bone remodeling. GSEA against hallmark gene sets indicated upregulation of mesenchymal phenotype and inflammatory response suggesting an underlying effect on the stroma driven by the presence of the JAK2VF clone. Qualitative x-ray of femurs indicated loss of bone density, suggesting disease-driven effect on the stroma (Fig B). Discussion: The development of our translatable model of MPN, where a small subset of cells drives the disease within unconditioned recipients represents an important milestone towards studying early disease pathology in MPNs. Using wt BMT, we demonstrated the successful establishment of long-term, low-level chimerism in unconditioned recipients. We found the clone-dependent impact on the stroma without myeloablative conditioning. Bulk transcriptomics and femur x-rays indicate aberrant bone morphology in BMT mice, despite the low level of engraftment (<5%) in the recipient system. Our data suggests that even low VAF of JAK2VF may support pathologic remodeling of the BM environment, warranting further exploration of patient BM niche early and late in the MPN course.

Glioblastoma (GBM) is the most common malignant primary brain tumor. GBM has an extremely poor prognosis and new treatments are badly needed. Efficient drug delivery to GBM is a major obstacle as the blood-brain barrier (BBB) prevents passage of the majority of cancer drugs into the brain. It is also recognized that the blood-brain tumor barrier (BTB) in the growing tumor represents a challenge. The BTB is heterogeneous and poorly characterized, but similar to the BBB it can prevent therapeutics from reaching effective intra-tumoral doses, dramatically hindering their potential. Here, we identified a 12-gene signature associated with the BTB, with functions related to vasculature development, morphogenesis and cell migration. We identified CDH5 as a core molecule in this set and confirmed its over-expression in GBM vasculature using spatial transcriptomics of GBM patient specimens. We found that the indirubin-derivative, 6-bromoindirubin acetoxime (BIA), could downregulate CDH5 and other BTB signature genes, causing endothelial barrier disruption in endothelial monolayers and BBB 3D spheroids in vitro. Treatment of tumor-bearing mice with BIA enabled increased intra-tumoral accumulation of the BBB non-penetrant chemotherapeutic drug cisplatin and potentiated cisplatin-mediated DNA damage by targeting DNA repair pathways. Finally, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved the efficacy of cisplatin in patient-derived GBM xenograms and prolonged their survival. Overall, our work reveals potential targets at the BTB for improved chemotherapy delivery and the bifunctional properties of BIA as a BTB modulator and potentiator of chemotherapy, supporting its further development.

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This “cell biopsy” method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Dysregulation of the adaptor protein Abelson interactor 1 (ABI1) is linked to malignant transformation. To interrogate the role of ABI1 in cancer development, we mapped the ABI1 interactome using proximity‐dependent labeling (PDL) with biotin followed by mass spectrometry. Using a novel PDL data filtering strategy, considering both peptide spectral matches and peak areas of detected peptides, we identified 212 ABI1 proximal interactors. These included WAVE2 complex components such as CYFIP1, NCKAP1, or WASF1, confirming the known role of ABI1 in the regulation of actin‐polymerization‐dependent processes. We also identified proteins associated with the TAK1‐IKK pathway, including TAK1, TAB2, and RIPK1, denoting a newly identified function of ABI1 in TAK1‐NF‐κB inflammatory signaling. Functional assays using TNFα‐stimulated, ABI1‐overexpressing or ABI1‐deficient cells showed effects on the TAK1‐NF‐kB pathway‐dependent signaling to RIPK1, with ABI1‐knockout cells being less susceptible to TNFα‐induced, RIPK1‐mediated, TAK1‐dependent apoptosis. In sum, our PDL‐based strategy enabled mapping of the ABI1 proximal interactome, thus revealing a previously unknown role of this adaptor protein in TAK1/RIPK1‐based regulation of cell death and survival.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

Abstract Understanding the distinct genetic features of the primary tumor and metastases in advanced prostate cancer (PCa) has clinical relevance and potential therapeutic implications. Studies utilizing differential gene expression analysis between primary tumors and sites of metastases continue to characterize the evolution of PCa from castration-sensitive (CSPC) through metastatic castration-resistant PCa (CRPC). This study aimed to investigate the genomic features of a cohort of patients (pts) with PCa with primary and/or metastatic tumors submitted for genomic analysis. We hypothesize that unique genomic features are associated with the site of metastases and can reveal potential site-specific drivers of disease progression as well as therapeutic targets. We performed a retrospective analysis of tumor biopsies by next-generation sequencing (NGS) from 45 patients with PCa. Targeted DNA sequencing of more than 500 cancer-associated genes was performed in paraffin-embedded tumor samples. All 45 tumors [(primary tumors (17), metastatic sites (28)] had DNA sequencing data available. Among these, 22 were additionally subjected to transcriptome profiling by RNA-seq. This subset comprised: 8 prostate and 14 metastatic samples [lymph node (7), bone (3), and visceral (4)]. Visceral sites included liver (1), colon (1), stomach (1), and bladder (1). The median age of pts was 66 years (range 50-84). The median prostate-specific antigen (PSA) level at diagnosis was 65 ng/mL (range 0.5-3,403). Among the 24 pts with Gleason score available, 17 (70%) had grade group 5 (3 - Gleason 10; 14 - Gleason 9). Primary tumors analyzed by NGS included CSPC (12) and CRPC (6). Most metastatic tumors were CSPC at biopsy (15 - CSPS; 12 - CRPC). Among the cohort, frequent gene alterations affected homologous repair deficiency genes (29.6%), as well as PTEN (20.3%) TMPRSS2 (25.9%), and FOXA1 (5%) genes. Differential gene expression and pathway analysis of RNA-seq data was performed using Bioconductor on the R statistical computing environment. Comparisons between primary and metastatic gene expression profiles revealed shared features among sample groups, including transcriptional changes to interferon-alpha and -gamma, cell cycle regulators and Myc targets, whereas others were distinct to the sites of metastasis. Bone metastases exhibited regulatory changes to JAK/STAT signaling and angiogenesis, as well as alterations in DNA repair. Visceral tumor samples were the most diverse cohort in terms of global transcriptome state, but were notable for MTOR and androgen signaling. Lymph node biopsies showed pronounced TNF-α signaling via NFkB and oxidative phosphorylation. Our results exemplify the cellular and molecular diversity between primary tumors and metastatic sites and highlight potential pathways and transcriptome profiles unique sites of metastases. Additional analysis on expanded cohorts of pts is ongoing. Citation Format: Luke B. Soliman, Andre L. De Souza, Praveen Srinivasan, Matthew Danish, Dragan J. Golijanin, Ali Amin, Anthony E. Mega, Wafik S. El-Deiry, Paul Bertone, Benedito A. Carneiro. Differential transcriptomic profiling of primary tumors and metastatic sites in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2195.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity.

The development of androgen resistance in advanced prostate cancer remains a challenging clinical problem. Because androgen deprivation therapy constitutes the backbone of first-line treatments for metastatic prostate cancer, the phenotypic switch from an androgen-dependent to an androgen-independent growth state limits the treatment options for these patients. This critical change from an androgen-dependent to an androgen-independent growth state can be regulated by the B-cell lymphoma gene 2 (BCL-2) family of apoptotic proteins. While the roles of BCL-2 protein family members in the carcinogenesis of prostate cancer have been well-studied, emerging data also delineates their modulation of disease progression to castration-resistant prostate cancer (CRPC). Over the past 2 decades, investigators have sought to describe the mechanisms that underpin this development at the molecular level, yet no recent literature has consolidated these findings in a dedicated review. As new classes of BCL-2 family inhibitors are finding indications for other cancer types, it is time to evaluate how such agents might find stable footing for the treatment of CRPC. Several trials to date have investigated BCL-2 inhibitors as therapeutic agents for CRPC. These therapies include selective BCL-2 inhibitors, pan-BCL-2 inhibitors, and novel inhibitors of MCL-1 and BCL-X L . This review details the research regarding the role of BCL-2 family members in the pathogenesis of prostate cancer and contextualizes these findings within the contemporary landscape of prostate cancer treatment.

Variation in DNA methylation between individuals has been shown to be influenced by both genetic and environmental factors. However, the relative impact of genetic and non-genetic factors on DNA methylation patterns across the mammalian genome has not been systematically studied. We performed whole-genome methylation analysis in two inbred mouse strains, revealing striking differences in the global distribution of DNA methylation. Although global methylation patterns were indistinguishable for most genomic features, a significant increase in the number of unmethylated CpG-island promoters and first exons was observed between strains. Experiments using F1 reciprocal hybrid strains demonstrated that the genotype of the mother dictated global DNA methylation patterns. Cross-fostering experiments ruled out a postnatal maternal effect on these differences and suggested that they were driven by a prenatal maternal effect, possibly via differential deposition of maternal gene products into the oocyte or uterine environment. These data demonstrate that maternal effects have a major impact on global DNA methylation patterns.

ABSTRACT Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and tethering of extracellular matrix (ECM) to substrates, making ECM tethering a potentially confounding variable in mechanical signalling investigations. Moreover, poor ECM tethering can lead to weak cell attachment. To address this, we developed StemBond hydrogels, a hydrogel formulation in which ECM tethering is stable and can be varied independently of stiffness. We show that soft StemBond hydrogels provide an optimal format for culturing embryonic stem (ES) cells. We find that soft StemBond substrates improve the homogeneity of ES cell populations, boost their self-renewal, and increase the efficiency of cellular reprogramming. Our findings underline how soft microenvironments impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions.

Several images were inadvertently duplicated in Fig. 1F and Fig. S1E. All the original data for these figures were reviewed by the journal and the correct panels are shown below.

The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.

Chromatin remodeling complexes play essential roles in metazoan development through widespread control of gene expression, but the precise molecular mechanisms by which they do this in vivo remain ill defined. Using an inducible system with fine temporal resolution, we show that the nucleosome remodeling and deacetylation (NuRD) complex controls chromatin architecture and the protein binding repertoire at regulatory regions during cell state transitions. This is primarily exerted through its nucleosome remodeling activity while deacetylation at H3K27 follows changes in gene expression. Additionally, NuRD activity influences association of RNA polymerase II at transcription start sites and subsequent nascent transcript production, thereby guiding the establishment of lineage-appropriate transcriptional programs. These findings provide a detailed molecular picture of genome-wide modulation of lineage-specific transcription by an essential chromatin remodeling complex as well as insight into the orchestration of molecular events involved in transcriptional transitions in vivo.

Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies provide transcriptome data from human preimplantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here we address these issues to assemble a complete gene expression time course spanning human preimplantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.