Biology subjects

Garifallou, J.

Publications and source records attributed to Garifallou, J..

7 recordsLinked to original sources

bioRxiv2026

Physiologic variation in sperm miRNAs tune embryonic gene regulatory programs and developmental outcomes

Small RNAs delivered by sperm can transmit environmentally regulated, epigenetically inherited phenotypes to offspring, yet the mechanisms by which modest changes in sperm microRNA abundance overcome dilution within the much larger egg to influence embryonic development remain unresolved. Here, we show that physiologically relevant variation in individual sperm miRNAs is sufficient to quantitatively program embryonic gene expression and developmental outcomes. Using parthenogenetic and fertilized embryos, we show that as few as 200 molecules of miR-200c-3p or miR-465c-3p induces reproducible, dose-dependent gene expression responses across defined developmental windows. Parthenogenetic embryos faithfully recapitulate early miRNA-driven gene expression changes observed in fertilized embryos, validating their use for isolating early regulatory mechanisms. We further developed AGO2-REMORA, an RNA adenosine base editor fused to Argonaute2 to map miRNA-mRNA interactions in embryos, revealing that early mRNA repression reflects direct miRNA targeting, while transcriptional changes at later stages arise as secondary consequences of these initial interactions. Furthermore, we show that modest elevation of miR-200c-3p during early development is sufficient to induce transcriptional alterations through early development and produce craniofacial phenotypes in late-stage embryos, recapitulating features of fetal alcohol syndrome associated with paternal alcohol consumption. Together, these findings establish a generalizable framework by which small perturbations in sperm miRNA content quantitatively modulate early gene regulatory programs, triggering cascades that persist throughout development and influence offspring phenotype.

developmental biology↗

bioRxiv2026

A single cell atlas defines perinatal factors that drive murine bone marrow development

Processes that direct initial colonization and maturation of the bone marrow remain elusive, despite lifelong importance to hematopoiesis. Bone marrow mesenchymal and stromal cell (BMSC) maturation establishes supportive niches prior to hematopoietic stem cell (HSC) recruitment. We define mouse BMSC progenitor identities and temporal emergence in a developmental single cell atlas spanning fetal life through 18 months of age. We clarify Cxcl12-abundant reticular (CAR) cell and osteoblast development, including temporal emergence of signaling modalities that direct HSC quiescence and regenerative capacity. CAR cells are absent until birth, with a developmental block during fetal maturation related to transcriptional changes during perinatal life. Temporal changes in Early B Cell Factor (Ebf) 1-3 expression and activity correlate with CAR formation and niche establishment, including Ebf2 repression and induction of Ebf3 activities that direct metabolic changes. Changes in systemic physiology underlie these transcriptional changes, including perinatal induction of lipid metabolism, inflammation, and hypoxic signaling. These systemic factors direct CAR cell emergence after birth, providing temporal and developmental resolution underlying the colonization and maturation of the bone marrow environment.

developmental biology↗

bioRxiv2025

Multimodal analysis of in vitro hematopoiesis reveals blood cell-specific genetic impacts on complex disease traits

In vitro hematopoiesis systems can be used to define mechanisms for blood cell formation and function, produce cell therapeutics, and model blood cell contributions to systemic disease. Hematopoietic progenitor cell (HPC) production remains inefficient, precluded by knowledge gaps related to specification and morphogenesis of specialized hemogenic endothelial cells, which undergo an endothelial-to-hematopoietic transition (EHT) to form HPCs. We elected to define changes in gene expression and chromatin organization during HPC formation to reveal regulatory mechanisms. Using paired single cell RNA/ATAC sequencing together with Hi-C, we profiled cells before and after EHT. Pathway analysis and pseudotime inferences confirmed a continuum of stromal and endothelial cells undergoing development into HE cells and lineage-based HPCs in vitro. In these cell types, we characterize cis-regulatory elements and transcriptional regulatory activities that facilitate EHT and HPC homeostasis, including for SNAI1, SOX17, TGF{beta}, STAT4, as well as for GFI1b and KLF1 in megakaryocyte- and erythroid-biased progenitors, respectively. We then leveraged our insights into chromatin organization among in vitro-derived cells to assess enrichments corresponding to human trait variation reported in human genome wide association studies. HPCs revealed locus enrichment for quantitative blood traits and autoimmune disease predisposition, which were particularly enriched in myeloid- and lymphoid-biased populations. Stromal and endothelial cells from our in vitro cultures were specifically enriched for accessible chromatin at blood pressure loci. Our findings reveal genes and mechanisms governing in vitro hematopoietic development and blood cell-related disease pathology.

genetics↗

bioRxiv2025

GM-CSF production by immune cells in steady state and autoimmune neuroinflammation mapped using fate reporting mice

The full GM-CSF expression spectrum in immune cells remains unclear, while CD4{square} T cells are the primary source. Using novel GM-CSF reporter/fate reporter transgenic mice, we tracked ongoing and past (YFP+) GM-CSF expression in various immune cells. GM-CSF was produced by diverse immune cells, including CD4+, CD8+, {gamma}{delta} T, NK, B, and CD11b+ cells, with expression patterns varying by cell type and organ with liver CD4+ T cells and NK cells showing the highest expression history in both naive and mice with EAE. GM-CSF expression was transient and permanently lost in most cells over time. In a mouse model of multiple sclerosis, effector memory CD4{square} T cells were the dominant CNS GM-CSF source, with higher expression than in other organs. CD4+YFP+ T cells, strongly expressing CXCR6, produced multiple cytokines. Transcriptomic analysis showed distinct gene expression profiles in effector memory CD4+ T cells compared to naive cells. YFP{square} Tregs represent functionally distinct subsets mirroring effector Th cells, expressing cytokines associated with Th lineages, especially during neuroinflammation. These findings identified distinct GM-CSF cellular sources across organs, highlighting a transient tissue microenvironment influence on GM-CSF production linked to CXCR6 expression.

immunology↗

bioRxiv2024

Integrated local and systemic communication factors regulate nascent hematopoietic progenitor escape during developmental hematopoiesis

Mammalian blood cells originate from specialized hemogenic endothelial (HE) cells in major arteries. During the endothelial-to-hematopoietic transition (EHT), nascent hematopoietic stem cells (HSCs) bud from the arterial endothelial wall and enter circulation, destined to colonize the fetal liver before ultimately migrating to the bone marrow. Mechanisms and processes that facilitate EHT and the release of nascent HSCs are incompletely understood, but may involve signaling from neighboring vascular endothelial cells, stromal support cells, circulating preformed hematopoietic cells, and/or systemic factors secreted by distal organs. In this study, we used single cell RNA sequencing analysis from human embryonic cells to identify relevant signaling pathways that support nascent HSC release. In addition to intercellular and secreted signaling modalities that have been previously functionally validated to support EHT and/or developmental hematopoiesis in model systems, we identify several novel modalities with plausible mechanisms to support EHT and HSC release. Our findings paint a portrait of the complex interregulated signals from the local niche, circulating hematopoietic/inflammatory cells, and distal fetal liver that support hematopoiesis.

developmental biology↗

bioRxiv2022

Human T follicular helper clones seed the germinal center-resident regulatory pool

How FOXP3+ T follicular regulatory (Tfr) cells simultaneously steer antibody formation toward microbe/vaccine recognition and away from self-reactivity remains unsettled. To explore human Tfr cell provenance, function and location heterogeneity, we used paired TCRVA/TCRVB sequencing to distinguish tonsillar Tfr cells clonally related to natural Tregs (nTfr) from those likely induced from Tfh cells (iTfr). The proteins iTfr and nTfr cells differentially expressed were utilized to pinpoint their in situ locations via multi-plex microscopy and establish divergent functional roles. In-silico and tonsil organoid tracking models corroborated the existence of separate Treg-to-nTfr and Tfh-to-iTfr developmental trajectories. In total, we have identified human iTfr cells as a distinct CD38-expressing, GC-resident, Tfh-descended subset that gains suppressive function while retaining capacities for B-cell help whereas CD38- nTfr cells are elite suppressors primarily localized to follicular mantles. Interventions differentially targeting Tfr subsets may provide therapeutic opportunities to boost immunity or more precisely treat autoimmune diseases. One sentence summaryHuman tonsillar Tfr clones descend from either Treg or Tfh lineages and provenance predicts their TCR repertoires, locations and functional characteristics.

immunology↗

bioRxiv2020

RUNX1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells: Mechanistic studies and drug correction

Patients with familial platelet disorder with a predisposition to myeloid malignancy (FPDMM) harbor germline monoallelic mutations in a key hematopoietic transcription factor RUNX1. Previous studies of FPDMM have focused on megakaryocyte (Mk) differentiation, and platelet production and signaling. However, the effects of RUNX1 haploinsufficiency on hematopoietic progenitor cells (HPCs) and subsequent megakaryopoiesis remains incomplete. To address this issue, we studied induced-pluripotent stem cell (iPSC)-derived HPCs (iHPCs) and Mks (iMks) from both patient-derived lines and a wildtype line modified to be RUNX1 haploinsufficient (RUNX1+/-), each compared to their isogenic wildtype control. All RUNX1+/- lines showed decreased iMk yield and depletion of a Mk-biased iHPC subpopulation. To investigate global and local gene expression changes underlying this iHPC shift, single-cell RNA sequencing was performed on sorted FPDMM and control iHPCs. We defined several cell subpopulations in FPDMM Mk-biased iHPCs. Analyses of gene sets upregulated in FPDMM iHPCs indicated enrichment for response to stress, regulation of signal transduction and response to cytokine gene sets. Immunoblotting studies in FPDMM iMks were consistent with these findings, but also identified augmented baseline c-Jun N-terminal kinase (JNK) phosphorylation, known to be activated by transforming growth factor {beta}1 and cellular stressors. J-IN8 and RepSox, small drugs targeting these pathways, corrected quantitative defects in FPDMM iHPC production. These findings were confirmed in adult human CD34+-derived stem and progenitor cells transduced with lentiviral RUNX1 short-hairpin (sh) RNA to mimic RUNX1+/-. These mechanistic studies of the defect in megakaryopoiesis in FPDMM suggest druggable pathways for clinical management of thrombocytopenia in affected patients. Key pointsO_LIRUNX1 haploinsufficiency results in a deficiency of megakaryocyte-biased hematopoietic progenitor cells (HPCs). C_LIO_LIRUNX1 haploinsufficiency elevates druggable proinflammatory and TGF{beta}R1-related pathways in HPCs. C_LI

molecular biology↗