Genes & Development

Co-transcriptional splicing is a conserved feature of eukaryotic gene expression. However, establishing the functional nature of this process has been difficult. Here using high throughput CRISPR/Cas9 screens we surprisingly find that SF3B3, the third largest subunit of the U2 snRNP complex, is a major regulator of RNA Pol II pause release and processivity. Remarkably, the absence of SF3B3 dramatically perturbs transcription but U2 snRNP assembly and RNA splicing remains unaffected. Mechanistically, SF3B3 coordinates the chromatin occupancy of transcriptional kinases (CDK9/12/13) alongside the PAF1c and Integrator complexes to regulate Pol II. Structure / function analyses of SF3B3 revealed that a metazoan specific 18aa sequence within its disordered tail phenocopies its loss and mediates the physical association and stability of SF3B5. We show that loss of SF3B5 mirrors SF3B3 deficiency suggesting this submodule, although resident within the U2 snRNP complex, evolved to primarily coordinate RNA Pol II in a splicing-independent manner.

SMC complexes influence virtually all DNA-dependent processes by organizing the genome via the process of loop extrusion. Although transcription has been implicated in regulating SMC complex function, the underlying mechanisms remain unclear. Further, the directionality of loop extrusion observed in biochemical experiments has been difficult to reconcile with the chromatin condensation observed in cells. Here, we use a quiescent yeast model to uncover the relationship between condensin loop extrusion and transcription. Condensin gradually relocates to transcribed gene promoters during quiescence entry, allowing us to dissect condensin targeting mechanisms temporally. Through targeted degradation experiments, we discover that topological stress generated by transcription leads to single-stranded DNA accumulation at promoters, and that these RPA-bound regions are loading sites and extrusion barriers for condensin. We further use a condensin mutant to determine that condensin extrudes loops asymmetrically in cells. We propose that antagonism by RPA universally regulates SMC complex function.

Germinal centers (GCs) support physiological B-cell mutagenesis and are considered lymphoma-permissive; nevertheless, lymphoma development is uncommon. Human in situ follicular neoplasia (ISFN) captures this paradox: premalignant B-cells can persist within GCs for prolonged periods without progressing to overt lymphoma. We found that human ISFN, but not normal GCs, are infiltrated by CD8 T-cells, suggesting that premalignant GC B-cells are locally immune-surveilled. Using mouse models that separate early premalignant fitness from lymphoma-associated evolution, we show that fitness-enhanced premalignant GC B-cells expand transiently but are selectively eliminated by infiltrating cytotoxic CD8 T-cells, while normal GC B-cells are spared. By contrast, evolved premalignant GC B-cells retain their fitness but disable productive CD8 T-cell cytotoxic differentiation, allowing persistence and lymphoma-like transcriptional and genomic evolution. These findings establish GCs as active immune-surveillance sites and show that progression from premalignancy to lymphoma requires both enhanced GC fitness and escape from local immune control. Key findingsGCs undergo active immune-surveillance to detect premalignant B-cells. Premalignant GC B-cells trigger cytotoxic CD8 T-cell responses. Lymphoma-associated evolution enables immune-escape within GCs. Fitness and immune-escape drive evolution from premalignancy to lymphoma. BlurbGerminal centers are considered lymphoma-permissive; however, progression from premalignancy is uncommon. Using models of human in situ follicular neoplasia, Zhang et al. demonstrate that infiltrating CD8 T-cells actively eliminate premalignant GC B-cells. Co-occurrence of lymphoma-like alterations blocks this cytotoxic T-cell response, driving immune escape and lymphoma evolution.

The three-dimensional (3D) genome architecture is highly elastic, adapting to nuclear processes such as transcription and the DNA damage response (Dekker & Mirny 2016; Carre-Simon & Fabre 2021). Nucleotide excision repair (NER) acts within this chromatin context to detect and repair mutagenic lesions induced by ultraviolet (UV) irradiation (Sancar 2016). UV irradiation has been shown to induce restructuring of the 3D genome across multiple scales, including chromatin compartments, domains, and loops. However, the extent to which NER activity contributes to this remodelling is unresolved, as the only prior study tracking such UV-induced changes was limited to repair-proficient cells (Kaya & Adebali 2025). Here, by combining genome-wide chromatin profiling of repair-deficient human cells with loop extrusion simulations, we show that lesion-stalled RNA polymerase II (RNAPII) and repair-associated barriers constrain loop extrusion. These events counter the loop-lengthening effects of UV-induced transcriptional shutdown, leading to shorter chromatin loops and reinforced chromatin domains that facilitate efficient lesion recognition and repair. The contribution by NER machinery underscores 3D genome reorganisation as an active mechanism both initiated and harnessed by DNA repair, rather than a passive consequence of DNA damage. The contribution by RNAPII extends its role beyond activating transcription-coupled repair to promoting a genome-wide repair-permissive state. Together, these findings advance our understanding of how nuclear processes coordinate on a shared chromatin substrate to preserve genome integrity.

During organogenesis, epithelial tissues undergo extensive three-dimensional (3D) remodeling while simultaneously generating specialized cell types. Whether these transient architectural states actively instruct lineage allocation remains unclear. Here we identify a morphogenetic stage in which resolution of epithelial stratification is required for lineage allocation and establishment of endocrine cell mass. We show that loss of the Hippo pathway regulator Merlin disrupts lumen morphogenesis and prevents formation of the transient 3D epithelial architecture that characterizes normal pancreas development. Failure to establish this architectural state alters lineage allocation, impairing acinar differentiation, markedly reducing adult endocrine cell mass, and disrupting glucose homeostasis. Mosaic analyses reveal that these lineage defects arise non-cell autonomously, demonstrating that epithelial architecture itself instructs cell fate decisions. Mechanistically, Merlin coordinates PI3K-regulated polarized membrane trafficking required for apical membrane biogenesis and lumen formation. Together, these findings identify Merlin-dependent membrane trafficking as a mechanism coupling epithelial morphogenesis to lineage allocation and demonstrate that transient developmental architectures can determine the cellular composition of mature organs.

The family of human G-patch proteins comprises more than 20 members, each characterized by a glycine-rich G-patch implicated in mediating interactions with RNA helicases. Here, we systematically identify the cognate RNA helicase of each G-patch protein, highlighting the association of DHX15 with a network of 20 G-patch cofactors. DHX35 and GPATCH1 represent a unique G-patch protein-RNA helicase pair, and we uncover a regulatory circuit between these partners. Comprehensive in vitro analyses of ATPase activity and RNA binding identify distinguishing features of DHX15- and non-DHX15-associated G-patch proteins, and demonstrate the roles of most G-patch proteins as bona fide stimulatory cofactors of DHX15. RNA interactome analyses of each G-patch protein and complementary transcriptome-wide alternative splicing analyses in cells lacking a G-patch protein reveal distinct modes of regulation of mRNA maturation by different G-patch proteins. For example, ZGPAT affects splicing indirectly through its requirement for efficient 2'-O-methylation of snRNAs, GPATCH8 exemplifies DHX15-associated alternative splicing modulation, whereas SUGP2 suppresses splicing in an RNA helicase-independent manner via direct binding to pre-mRNA introns.

Premature termination of transcription (PTT), also known as attenuation, is a conserved gene regulatory mechanism that operates across all domains of life and in viruses. Attenuation enables rapid cellular responses to environmental and metabolic changes and fine-tunes expression of biosynthetic genes. In Saccharomyces cerevisiae, attenuation of RNA Polymerase II (Pol II) transcription was first linked to the Nrd1-Nab3-Sen1 (NNS) termination pathway for non-coding RNAs, and the mRNA 3-end processing factor Hrp1 has been implicated more recently. Substitutions in Hrp1 RNA Recognition Motifs (RRMs) cause attenuator readthrough and reduce RNA-binding affinity in vitro, but direct evidence for Hrp1 functioning at attenuators in vivo remains limited. Here, we characterized 5-end RNA terminator elements from several genes, including RAD3, SNG1, MNR2, and CPR8. Readthrough mutations clustered in AU-rich regions resembling polyadenylation site (pA) efficiency elements, consistent with Hrp1 binding targets. Amino acid substitutions of Hrp1 RRM residue F162 revealed a general requirement for aromaticity in RNA recognition that varied to some degree by gene context. To test Hrp1-RNA interactions independent of other yeast factors, we adapted a bacterial 3-hybrid (B3H) assay. Hrp1 interacted with RNA derived from the GAL7 3-end pA site and 5-end terminator regions of RAD3, MNR2, and CPR8. Mutations in AU-rich RNA regions that disrupted Pol II attenuation in yeast generally impaired B3H interactions. However, some Hrp1 mutants (M191T, I270T, D271G, M275V, T280I) retained binding to CPR8 terminator RNA, suggesting their defects require additional yeast components. These results demonstrate that Hrp1 is sufficient to bind multiple UA-rich attenuator RNAs in vivo, expanding Hrp1 function to include early transcription events.

Distinct molecular variants of the brain cancer ependymoma are distributed along the rostral-caudal extent of the central nervous system (CNS). Historically proposed to arise from ventricular ependyma, recent studies have suggested conflicting cellular origins, including the neural radial glia and the roof plate lineages. Using single-cell transcriptomics, immunohistochemistry, and lineage tracing, we demonstrate that ependymomas across all CNS compartments transcriptionally mirror MSX1+ve pre-neural crest/roof plate (Pre-NC/RP) lineage derivatives. Ependymoma subgroups recapitulate the spatial and molecular diversity of regional Pre-NC/RP populations, while retaining conserved MSX1 expression. Expression of the oncogenic fusion ZFTA-RELA within the murine Pre-NC/RP lineage generated tumors that faithfully resembled human ependymoma. These findings identify a common embryonic cellular origin for ependymomas and reconcile previously conflicting models of tumorigenesis.

Macrophages are innate immune cells of embryonic or adult origin with tissue specific roles in homeostasis, disease surveillance, and wound repair that can be co-opted to promote tumor growth and spread1-11. An understanding of the specific roles of macrophage subsets in lung tumor initiation and progression could promote new therapeutic approaches for this deadly disease. Here, we show that KRASG12D mutations in lung epithelium drive proliferation of resident, embryonically-derived alveolar macrophages, which then promote tumor cell proliferation and protection from ferroptosis, leading to tumor progression. Using genetically engineered mouse models of mutant KRASG12D non-small cell lung cancer12,13, we found that alveolar macrophages accumulate by proliferation in response to tumor cell-secreted IL-34, recapitulating events observed in late embryonic lung development. Tumor alveolar macrophages in turn drive IGF-1-dependent tumor cell proliferation. Neutralization or deletion of IL-34 suppresses IGF-1 expression, reduces macrophage and tumor cell proliferation and inhibits tumor progression. High IL34 and IGF1 correlate with poor survival in KRASG12D/V lung adenocarcinomas and in other solid tumors, indicating that bi-directional proliferative signaling between resident macrophages and tumor cells can drive human lung tumor progression. These studies identify resident macrophage-tumor cell interactions as key interception points for lung cancer therapy.

Pathologic white adipose tissue (WAT) remodeling, characterized by fibrosis, inflammation, and adipocyte dysfunction, is a hallmark and driver of metabolic disease in obesity1. Here, we show that legacy effects of early physiological or pharmacological interventions driving adaptive adipose remodeling can mitigate maladaptive WAT remodeling and metabolic dysfunction when developing obesity later in life. Cold exposure or beta3-adrenergic receptor (beta3AR) agonism (CL316,243) induced thermogenic remodeling of WAT in male mice. After a prolonged recovery at room temperature, trained epididymal WAT reverted to an energy-storing state but retained a population of adipocytes resembling metabolically flexible visceral adipocytes found in human metabolically healthy obesity. The legacy of the antecedent treatment conferred lasting protection against glucose intolerance when later developing high fat diet (HFD)-induced obesity, with insulin sensitivity persisting for at least 20 weeks of overnutrition. This metabolic resilience was accompanied by healthy epididymal WAT expansion with reduced fibrosis and inflammation. Our findings demonstrate that short-term interventions, without genetic manipulation, can train adipose tissue, enhancing its long-term plasticity and conferring durable protection against future obesity-associated insulin resistance.

Insulin is an essential regulator of glucose homeostasis in vertebrates, and impairment of its synthesis or action leads to diabetes with severe health complications in humans. It is therefore essential to understand how beta cells control insulin synthesis and secretion, including the transcription, translation and decay of its messenger RNA. Using sequencing-based poly(A) tail length profiling from human tissue, genetic evidence for type 2 diabetes, bulk and single-cell transcriptomics and perturbation experiments, here we find that the insulin mRNA is stabilized by the activity of noncanonical poly(A) polymerases of the Tent5 family. We show that Tent5 activity is specific, promoted by both localization at the endoplasmic reticulum and regulatory sequences within the insulin mRNA and regulated by glucose. Overall, our findings provide a mechanistic link between the dynamic control of insulin production by beta cells and the direct regulation of insulin mRNA metabolism.

BACKGROUNDTAR DNA binding protein - 43 (TDP-43) nuclear loss is a pathological hallmark of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and related neurodegenerative disorders. While the consequences of TDP-43 dysfunction have been well-characterized, the mechanisms driving TDP-43 mislocalization remain poorly understood. Previous observations of altered localization and function of the adenosine-to-inosine (A-to-I) RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) in ALS/FTD tissue prompted us to investigate whether dysregulated RNA editing contributes to pathological TDP-43 nucleocytoplasmic trafficking. METHODSTDP-43 cytoplasmic mislocalization was assessed following ADAR2 and TDP-43 co-overexpression in HEK293T cells and a Drosophila model co-overexpressing human TDP-43 and dADAR in motor neurons. We further evaluated TDP-43 mislocalization through both HeLa cell assays and interspecies heterokaryon assays. Next, we assessed TDP-43 binding to A-to-I edited RNA oligomers through electrophoretic mobility shift assays (EMSAs), and investigated inosine-containing RNAs in vivo via TDP-43 RNA immunoprecipitation followed by sequencing (RIP-seq) datasets from human TDP-43-expressing Drosophila. Finally, RNAseq and enhanced cross-linking and immunoprecipitation (eCLIP-seq) were performed in SH-SY5Y cells overexpressing three ADAR2 variants with differing editing activity to identify editing-related transcriptional alterations and RNAs differentially bound to TDP-43. RESULTSADAR2 overexpression reduced the nucleocytoplasmic (N:C) ratio of TDP-43 in HEK293T cells in a ADAR2 catalytic activity- and TDP-43 RNA-binding capacity-dependent manner. Drosophila motor neurons overexpressing dADAR also exhibited decreased nuclear TDP-43. Interspecies heterokaryons and permeabilized HeLa cell assays demonstrated that catalytically active ADAR2 and synthetic inosine-containing RNA oligomers, respectively, enhance nuclear export of endogenous TDP-43. EMSAs revealed preferential binding of TDP-43 to inosine-containing RNAs relative to unedited RNAs, and analysis of Drosophila RIP-seq datasets demonstrated enrichment of edited transcripts within TDP-43-bound RNAs. Finally, RNAseq and eCLIP-seq analyses identified editing-dependent alterations in gene expression and TDP-43 RNA-binding profiles in SH-SY5Y cells overexpressing active ADAR2 variants. CONCLUSIONSTogether, our findings identify A-to-I RNA editing as a previously unrecognized regulator of TDP-43 localization and RNA interactions. These results support a model where altered RNA editing modifies TDP-43-RNA interactions, promoting increased nuclear export of TDP-43. Broadly, our work highlights RNA editing dysregulation as a potential contributor to early pathogenic mechanisms underlying TDP-43 proteinopathies.

Rhabdoid tumors are very aggressive rare pediatric cancers with poor survival affecting very young children. They are characterized by the bi-allelic loss of SMARCB1 or SMARCA4, which is suspected to occur prenatally. However, their genomic evolution is not well understood. Here we assembled the largest cohort of whole-genome sequenced rhabdoid tumors to date, comprising 97 tumors from 88 children. We discovered that, in 42% of cases, the bi-allelic inactivation of the driver gene occurred via a Copy Number Neutral-Loss of Heterozygosity (CN-LOH). We exploited these CN-LOH events and the steady accumulation of age-related mutations in the tumor genomes to estimate the age of donors at the time of occurrence of the driver event and at the time of emergence of the clonal expansion. Across all cases with CN-LOH, the loss of the driver gene occurred very early during prenatal development. However, the clonal expansion that ultimately gave rise to the tumor occurred at different times during infancy, even several years after the acquisition of the founder event. These results indicate that probably other factors, besides the genetic driver event, are required to promote rhabdoid tumorigenesis.

HSP90/R2TP is an essential quaternary chaperone composed of RPAP3, PIH1D1 and the RUVBL1/RUVBL2 AAA+ ATPases. These enzymes are also part of the INO80, SRCAP and TIP60 complexes, but the relationship between these chromatin remodelers and R2TP remains unclear. Here, we performed systematic analyses of the R2TP-specific subunits RPAP3 and PIH1D1. We validated 115 interaction partners and found that many were sensitive to HSP90 or R2TP inhibition. In yeast, epistatic screens revealed functional interactions with Ino80, Swr1 (SRCAP) and NuA4 (TIP60). Consistently, human RPAP3 physically interacted with subunits of INO80, SRCAP and TIP60 and was required for the formation of these complexes. More specifically, RPAP3 enabled the co-translational association of RUVBL1/RUVBL2 with the motor subunit of these chromatin remodelers. In vitro, the client-binding domain of RUVBL1/RUVBL2 modulated their interaction with RPAP3, suggesting that client subunits displace RPAP3 from nascent complexes. Thus, R2TP is an early chaperone of TIP60, SRCAP and INO80, which leaves RUVBL1/RUVBL2 as resident scaffolding subunits.

Hypermutational processes, including those driven by the APOBEC3 family of cytidine deaminases, generate abundant neoantigens yet give rise to tumors that evade immune recognition. Here, using multi-omics analyses followed by functional validation, we identified a tumor-intrinsic immune-cloaking mechanism in neoantigen-rich epithelial cancers, characterized by coordinated suppression of antigen presentation, immune-recruiting cytokines and immune-checkpoint programs. In bladder cancer, genome-wide copy-number analysis identified recurrent 1q23.3 amplification as a genomic feature of a neoantigen-high/CD8-low tumor state. Within this locus, NECTIN4 emerged as the dominant candidate effector, outperforming extrachromosomal DNA status as a predictor of immune-neoantigen discordance. Similar associations were observed across breast and lung cancers. Functional studies demonstrated that NECTIN4 was sufficient to establish a T-cell-poor tumor microenvironment and confer resistance to PD-1 blockade in immunocompetent mice. Mechanistically, NECTIN4 engaged a DDR1-SHP2 axis that suppressed STAT1 phosphorylation, silencing tumor-cell immune-engagement programs. NECTIN4 blockade restored STAT1 activity and reduced tumor growth, indicating that the cloaked state is pharmacologically reversible. Mutational signature, breakpoint motif, timing and clonality analyses, together with APOBEC3B expression and germline genetic evidence, linked APOBEC3-mediated mutagenesis to recurrent 1q23.3 amplification encompassing NECTIN4. These findings reveal how neoantigen-generating mutational processes can be coupled to structural genome evolution to enable tumor-intrinsic immune cloaking through a therapeutically targetable NECTIN4-DDR1-SHP2 axis.

Embryonic development is marked by the successive restriction of developmental potential and the specification of embryonic and extraembryonic lineages. Yet, how these lineage decisions are established, and how the epigenome is remodelled to promote and restrict cell fate transitions, remains poorly understood. Here, we demonstrate that SALL2 knockdown in primed human pluripotent stem cells (hPSCs) triggers a trophectoderm (TE)-like phenotype, characterized by palisade-like morphology and the up-regulation of TE-associated genes. Mechanistically, SALL2 physically interacts with the key TE driver TEAD4 and maintains bivalent, repressive chromatin (H3K4me3/H3K27me3) at TE-specific loci. Reduced SALL2 led to enhanced TEAD4 occupancy and disrupted H3K27me3 and increased active chromatin marks at TE genes. Importantly, depletion of TEAD4 abolished the TE-like phenotype induced by SALL2 knockdown, demonstrating that TEAD4 is required for the downstream effects of SALL2 loss. In support of this, blastoid-like aggregates can be generated from primed hPSCs with SALL2 knocked down. Together, our findings identify SALL2 as a key epigenetic barrier that restrains TE lineage commitment by limiting TEAD4-dependent activation of the trophoblast transcriptional program.

Tumour progression relies on reciprocal communication between genetically altered cancer cells and surrounding stromal cells. While the genetic alterations that initiate tumorigenesis have been extensively studied, the dysregulated feedback signalling provided by co-opted stromal cells remains poorly understood. Here, we used a Drosophila cancer model to address this question and identified the matricellular protein SPARC as a mediator of tumour-stroma communication. SPARC is produced by mesenchymal cells and transferred into epithelial tumour cells, where it is internalized through the endocytic pathway. Following uptake, SPARC accumulates in Rab7 positive late endosomes and colocalize with the Notch ligand Delta. SPARC internalization promotes endosomal enlargement and reduces endosome dynamics. Increased SPARC levels in epithelial tumours indirectly attenuate Notch signalling activity through at least altered Delta trafficking. We further identify the N-terminal acidic domain of SPARC as specifically required for its targeting to Delta-associated endosomes. Together, our findings uncover a stromal feedback mechanism by which SPARC modulates Notch signalling through endosomal regulation during tumour development.

Epithelial cells can encode prior damage into lasting epigenetic and functional states, enabling a primed response to future insults. In the pancreas, acute injury induces reversible acinar cell reprogramming toward a progenitor-like identity that persists beyond repair, supporting resilience to recurrent injury but creating a permissive state for malignant transformation. Given the central role of the tissue niche in stem cell regulation, we investigated microenvironmental adaptations that sustain this primed epithelial state. Using genetic mouse models and ex vivo organoid co-cultures, we identify a sex-specific sensory neural memory after pancreatitis that sustains long-term epithelial plasticity through a CGRP-dependent neuron-epithelial axis. We show that sex differences in acute inflammation drive neutrophil-dependent suppression of neural activation in females, decoupling neural memory from epithelial plasticity after repair. In males, neural memory promotes post-injury plasticity, revealing tissue memory as coordinated adaptation between epithelial progenitors and their niche.

The proneural transcription factor ASCL1 is well established as a pioneering neuronal reprogramming factor in several contexts. However, the extent to which distinct tissue types adopt a similar or divergent response to the same ASCL1 fate challenge is unclear. By expressing ASCL1 across germ layers and tissues of the developing Xenopus neurula embryo, we reveal widespread but largely distinct transcriptomic and chromatin responses to ASCL1. Ectopic ASCL1 can access partially open chromatin sites across all tissues, but it is able to further open those sites specifically in neuroectoderm. Using motif enrichment and accessibility analyses we identify Sox transcription factor motifs as enriched and preferentially accessible at ASCL1-bound regions in neuroectoderm. We show that co-expression of Sox3 and ASCL1 can enable the activation of otherwise refractory ASCL1 target genes in mesoderm and epidermal skin. Our findings reveal how the chromatin landscape and co-factor availability work together to modulate the response to transcription factor-driven fate challenge during development. Summary statementSingle-cell multi-omics reveals how chromatin accessibility and cofactor availability constrains ASCL1-driven neuronal reprogramming across tissues in the developing Xenopus embryo.

Mechanical forces from blood flow are essential for production of hematopoietic stem and progenitor cells (HSPCs) during embryogenesis, but the molecular mechanisms by which hemodynamic cues are sensed and orchestrate endothelial-to-hematopoietic (EHT) transition remain incompletely defined. We previously identified YAP mechanotransduction as a key integrator of physical forces with EHT. Here we show that hemodynamic forces can activate YAP signaling via the mechanoresponsive ion channel Piezo1 in human iPSC-derived hemogenic endothelium (HE) and zebrafish embryos. Investigation of the Piezo1/YAP axis revealed shared and unique roles of YAP and its paralogue TAZ in EHT. Mechanistically, we find a requirement for the Tead DNA-binding co-factor in YAP/TAZ-dependent control of HSPC number, and note that TAZ uniquely augments transcriptional output of the hematopoietic master regulator Runx1 via direct protein-protein interactions. By comprehensive scRNA-sequencing of YAP/TAZ gain-of-function (GOF) and yap-deficient cells from zebrafish, we reveal that YAP/TAZ promotes HSC production by positively regulating gene programs for hematopoietic self-renewal, cell cycle, and glycolysis-to-oxidative phosphorylation switching, while preventing reversion to endothelial identity. Importantly, comparison of GOF transcriptomes and functional analyses suggest decoupling of metabolic/proliferative and endothelial gene regulatory modules between YAP and TAZ: while either can functionally compensate for loss of the other in EHT, indiscriminate overactivation of TAZ enhances an endothelial program over pro-hematopoietic fate, ultimately blunting progression of HSPC production. Given that hemodynamic cues are integrated simultaneously by arterial and HE cells in embryonic vessels in which EHT occurs, these findings have strong implications for strategies designed to introduce biomechanical cues to in vitro hematopoietic differentiation systems to drive HSC production.