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The stress-activated protein kinases (SAPK) and a group of dual-specificity kinases with potential roles in the control of apoptosis and proliferation. In most cells they are regulated through phosphorylation by MKK-4. We have investigated the role of MKK-4 in T cell development and function by generating transgenic animals expressing catalytically inactive MKK-4 (dMKK-4) in the thymus. Our results show that overexpression of dMKK-4 does not interfere with normal T cell development. Furthermore, expression of dMKK-4 inhibits Fas- but not phorbol ester plus ionomycin-induced activation of SAPK, suggesting that a SAPK kinase different from MKK-4 is responsible for the regulation of SAPK activation after stimulation of T cells with phorbol ester plus ionomycin. We then analyzed the effect of dMKK-4 of Fas-induced apoptosis of thymocytes. Our results show that activation of SAPK is not a necessary even in Fas-induced apoptosis of thymocytes. [ABSTRACT FROM PUBLISHER]

Elucidating mechanisms of T cell development can guide in vitro T cell differentiation from induced pluripotent stem cells (iPSCs) and facilitate off-the-shelf T cell-based immunotherapies. Using a stroma-free human iPSC-T cell differentiation platform, we screened for epigenetic modulators that influence T cell specification and identified the H3K9-directed histone methyltransferases G9a/GLP as repressors of T cell fate. We show that G9a/GLP inhibition during specific time windows of differentiation of hematopoietic stem and progenitor cells (HSPCs) skews cell fates toward lymphoid lineages. Inhibition of G9a/GLP promotes the production of lymphoid cells during zebrafish embryonic hematopoiesis, demonstrating the evolutionary conservation of G9a/GLP function. Importantly, chemical inhibition of G9a/GLP facilitates the generation of mature iPSC-T cells that bear transcriptional similarity to peripheral blood αβ T cells. When engineered to express chimeric antigen receptors, the epigenetically engineered iPSC-T cells exhibit enhanced effector functions in vitro and durable, persistent antitumor activity in a xenograft tumor-rechallenge model., Competing Interests: Declaration of interests R.J., G.Q.D., and Boston Children’s Hospital hold intellectual property relevant to the generation of iPSC-derived T cells. T.M.S. has received sponsored research support from Elevate Bio. G.Q.D. is a member of Cell Stem Cell’s advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The field cancerization theory suggests that a group of cells containing oncogenic mutations are predisposed to transformation 1, 2 . We previously identified single cells in BRAF V600E ;p53 -/- zebrafish that reactivate an embryonic neural crest state before initiating melanoma 3-5 . Here we show that single cells reactivate the neural crest fate from within large fields of adjacent abnormal melanocytes, which we term the "cancer precursor zone." These cancer precursor zone melanocytes have an aberrant morphology, dysplastic nuclei, and altered gene expression. Using single cell RNA-seq and ATAC-seq, we defined a distinct transcriptional cell attractor state for cancer precursor zones and validated the stage-specific gene expression initiation signatures in human melanoma. We identify the cancer precursor zone driver, ID1, which binds to TCF12 and inhibits downstream targets important for the maintenance of melanocyte morphology and cell cycle control. Examination of patient samples revealed precursor melanocytes expressing ID1, often surrounding invasive melanoma, indicating a role for ID1 in early melanomagenesis. This work reveals a surprising field effect of melanoma initiation in vivo in which tumors arise from within a zone of morphologically distinct, but clinically covert, precursors with altered transcriptional fate. Our studies identify novel targets that could improve early diagnosis and prevention of melanoma.

Blood development and regeneration require rapid turnover of cells, and ribonucleic acid (RNA) modifications play a key role in it via regulating stemness and cell fate regulation. RNA modifications affect gene activity via posttranscriptional and translation-mediated mechanisms. Diverse molecular players involved in RNA-modification processes are abundantly expressed by hematopoietic stem cells and lineages. Close to 150 RNA chemical modifications have been reported, but only N6-methyl adenosine (m 6 A), inosine (I), pseudouridine (Ψ), and m1A-a handful-have been studied in-cell fate regulation. The role of RNA modification in blood diseases and disorders is an emerging field and offers potential for therapeutic interventions. Knowledge of RNA-modification and enzymatic activities could be used to design therapies in the future. Here, we summarized the recent advances in RNA modification and the epitranscriptome field and discussed their regulation of blood development and regeneration., Competing Interests: Conflict of Interest Disclosure Dr LI Zon is a Howard Hughes Medical Institute investigator. The other author does not have any conflicts of interest to declare in relation to this work., (Published by Elsevier Inc.)

Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8 + T cells in the tumor. In zebrafish, CD8 + T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8 + T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response., Competing Interests: L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, Scholar Rock, and Branch Biosciences. He is a consultant for Celularity and Cellarity. JVA is on the BMS and AstraZeneca advisory board and a consultrant for MSD, Janssen. F.S.H reports grants and personal fees from Bristol-Myers Squibb, personal fees from Merck, grants and personal fees from Novartis, personal fees from Surface, personal fees from Compass Therapeutics, personal fees from Apricity, personal fees from 7 Hills Pharma, personal fees from Bicara, personal fees from Checkpoint Therapeutics, personal fees from Bioentre, personal fees from Gossamer, personal fees from Iovance, personal fees from Catalym, personal fees from Immunocore, personal fees from Kairos, personal fees from Rheos, personal fees from Zumutor, personal fees from Corner Therapeuitcs, personal fees from Puretech, personal fees from Curis, personal fees from Astra Zeneca, personal fees from Solu Therapeutics, outside the submitted work; In addition, Dr. Hodi has a patent Methods for Treating MICA-Related Disorders (#20100111973) with royalties paid, a patent Tumor antigens and uses thereof (#7250291) issued, a patent Angiopoiten-2 Biomarkers Predictive of Anti-immune checkpoint response (#20170248603) pending, a patent Compositions and Methods for Identification, Assessment, Prevention, and Treatment of Melanoma using PD-L1 Isoforms (#20160340407) pending, a patent Therapeutic peptides (#20160046716) pending, a patent Therapeutic Peptides (#20140004112) pending, a patent Therapeutic Peptides (#20170022275) pending, a patent Therapeutic Peptides (#20170008962) pending, a patent THERAPEUTIC PEPTIDES Ðherapeutic PeptidesÐatent number: 9402905 issued, a patent METHODS OF USING PEMBROLIZUMAB AND TREBANANIB pending, a patent Vaccine compositions and methods for restoring NKG2D pathway function against cancers Ðatent number: 10279021 issued, a patent Úntibodies that bind to MHC class I polypeptide-related Sequence A Ð’dÐatent number: 10106611 issued, a patent ÚNTI-GALECTIN ANTIBODY BIOMARKERS PREDICTIVE OF ANTI-IMMUNE CHECKPOINT AND ANTI-ANGIOGENESIS RESPONSES Ð’dÐublication number: 20170343552 pending, and a patent Antibodies against EDIL3 and methods of use thereof pending. JVA: Advisory board: BMS, AstraZeneca Consultant: MSD, Janssen.

Hematopoietic stem cells are regulated by endothelial and mesenchymal stromal cells in the marrow niche1-3. Leukemogenesis was long believed to be solely driven by genetic perturbations in hematopoietic cells but introduction of genetic mutations in the microenvironment demonstrated the ability of niche cells to drive disease progression4-8. The mechanisms by which the stem cell niche induces leukemia remain poorly understood. Here, using cellular barcoding in zebrafish, we found that clones of niche endothelial and stromal cells are significantly expanded in leukemic marrows. The pro-angiogenic peptide apelin secreted by leukemic cells induced sinusoidal endothelial cell clonal selection and transcriptional reprogramming towards an angiogenic state to promote leukemogenesis in vivo. Overexpression of apelin in normal hematopoietic stem cells led to clonal amplification of the niche endothelial cells and promotes clonal dominance of blood cells. Knock-out of apelin in leukemic zebrafish resulted in a significant reduction in disease progression. Our results demonstrate that leukemic cells remodel the clonal and transcriptional landscape of the marrow niche to promote leukemogenesis and provide a potential therapeutic opportunity for anti-apelin treatment.

Macrophages maintain hematopoietic stem cell (HSC) quality by assessing cell surface Calreticulin (Calr), an "eat-me" signal induced by reactive oxygen species (ROS). Using zebrafish genetics, we identified Beta-2-microglobulin (B2m) as a crucial "don't eat-me" signal on blood stem cells. A chemical screen revealed inducers of surface Calr that promoted HSC proliferation without triggering ROS or macrophage clearance. Whole-genome CRISPR-Cas9 screening showed that Toll-like receptor 3 (Tlr3) signaling regulated b2m expression. Targeting b2m or tlr3 reduced the HSC clonality. Elevated B2m levels correlated with high expression of repetitive element (RE) transcripts. Overall, our data suggest that RE-associated double-stranded RNA could interact with TLR3 to stimulate surface expression of B2m on hematopoietic stem and progenitor cells. These findings suggest that the balance of Calr and B2m regulates macrophage-HSC interactions and defines hematopoietic clonality.

Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cyclic AMP (cAMP) response elements (CREs) in humans, mice, and fish drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for early B cell factor (Ebf/EBF) transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work identifies the functional conservation of two transcription factors in promoting tendon fate., Competing Interests: Declaration of interests L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, and Scholar Rock and is a consultant for Celularity., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein ( TIE:EGFP ). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP + melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2 , a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP + regions and preferentially phagocytose TIE:EGFP + melanoma cells compared to TIE:EGFP - melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors., Competing Interests: HN, AT, JB, MX, CB, SY, KK, AM No competing interests declared, LZ L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, Scholar Rock, and Branch Biosciences. He is a consultant for Celularity and Cellarity, (© 2024, Noonan, Thornock et al.)

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

Hematopoietic stem cell transplantation requires the collection of hematopoietic cells from patients or stem cell donors. Granulocyte colony-stimulating factor (G-CSF) is widely used in the clinic to mobilize hematopoietic stem and progenitor cells (HSPCs) from the adult bone marrow niche into circulation, allowing a collection of HSPCs from the blood. The mechanism by which G-CSF acts to mobilize HSPCs is unclear, with some studies showing a direct stimulation of stem cells and others suggesting that myeloid cells are required. In this study, we developed a heat-inducible G-CSF transgenic zebrafish line to study HSPC mobilization in vivo. Live imaging of HSPCs after G-CSF induction revealed an increase in circulating HSPCs, demonstrating a successful HSPC mobilization. These mobilized HSPCs went on to prematurely colonize the kidney marrow, the adult zebrafish hematopoietic niche. We eliminated neutrophils or macrophages using a nitroreductase-based cell ablation system and found that G-CSF still mobilizes HSPCs from the niche. Our findings indicate that neutrophils and macrophages are not required for G-CSF-induced HSPC mobilization from the embryonic hematopoietic niche., Competing Interests: Conflict of Interest Disclosure L.I.Z. is a founder and stockholder in Fate Therapeutics, Scholar Rock, Camp 4, Triveni, and Branch Bio. He is also a stockholder and consultant to Cellarity and Caper Bio. All other authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2024 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with a binding preference for G-quadruplex RNA. In this work, we present a 3.3-Å-resolution cryo-electron microscopy structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface composed of two copies of the catalytic subunit EZH2, thereby blocking nucleosome DNA interaction and histone H3 tail accessibility. Furthermore, an RNA-binding loop of EZH2 facilitates the handoff between RNA and DNA, another activity implicated in PRC2 regulation by RNA. We identified a gain-of-function mutation in this loop that activates PRC2 in zebrafish. Our results reveal mechanisms for RNA-mediated regulation of a chromatin-modifying enzyme.

Acquired genetic or cytogenetic alterations in a blood stem cell that confer clonal fitness promote its relative expansion leading to clonal hematopoiesis (CH). Despite a largely intact hematopoietic output, CH is associated with a heightened risk of progression to hematologic malignancies and with non-hematologic health manifestations, including cardiovascular disease and overall mortality. We focus on the evidence for the role of inflammation in establishing, maintaining and reciprocally being affected by CH. We describe the known pro-inflammatory signals associated with CH and preclinical studies that elucidated the cellular mechanisms involved. We review the evolving literature on early-onset CH in germline predisposition conditions and the possible role of immune dysregulation in this context., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function., Competing Interests: Declaration of interests R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, Dewpoint Therapeutics, and Paratus Sciences. R.A.Y. is a member of Molecular Cell’s advisory board. O.O. and J.E.H. are consultants at Camp4 Therapeutics. L.I.Z. is a founder and stockholder of Fate Therapeutics, Camp4 Therapeutics, Amagma Therapeutics, Scholar Rock, and Branch Biosciences. L.I.Z. is a consultant for Celularity and Cellarity. The Whitehead Institute has filed a patent application related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied., Results: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells., Conclusions: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche., Competing Interests: Declaration of interests L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, and Scholar Rock. He is a consultant for Celularity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate., Competing Interests: Declaration of interests L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, Scholar Rock, and Branch Biosciences. He is a consultant for Celularity and Cellarity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Prostaglandin E2 (PGE 2 ) and 16,16-dimethyl-PGE 2 (dmPGE 2 ) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122 , 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8 , 445-458 (2011); W. Goessling et al. Cell 136 , 1136-1147 (2009)]. Here, we report that PGE 2 -induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE 2 -inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE 2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE 2 , lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species., Competing Interests: Declaration of interests A.J.W. is a member of the scientific advisory board for Frequency Therapeutics, a company that aims to develop medicines to target endogenous stem cells to restore regenerative capacity; a consultant for Kate Therapeutics, a company that seeks to develop genetic medicines for muscle diseases; and a co-founder of Elevian, Inc., a company aimed at preserving the health of older individuals by targeting blood-borne geroproteins. L.I.Z. and A.J.W. are inventors on patent applications filed through Harvard University and Children’s Hospital regarding myogenic specification of embryonic cells from fish and mammals, and S.T., L.I.Z., and A.J.W. are inventors on patent applications filed through Harvard University regarding chemical modifiers of muscle engraftment by myogenic precursors. L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, and Scholar Rock. He is a consultant for Celularity., (Published by Elsevier Inc.)

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 ( CDK13 ) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.

Cancer genetics has uncovered many tumor-suppressor and oncogenic pathways, but few alterations have revealed mechanisms involved in tumor spreading. Here, we examined the role of the third most significant chromosomal deletion in human melanoma that inactivates the adherens junction gene NECTIN1 in 55% of cases. We found that NECTIN1 loss stimulates melanoma cell migration in vitro and spreading in vivo in both zebrafish and human tumors specifically in response to decreased IGF1 signaling. In human melanoma biopsy specimens, adherens junctions were seen exclusively in areas with low IGF1 levels, but not in NECTIN1-deficient tumors. Our study establishes NECTIN1 as a major determinant of melanoma dissemination and uncovers a genetic control of the response to microenvironmental signals., (© 2022. The Author(s).)

The zebrafish (Danio rerio) is among the most widely used model animals in scientific research. Historically, these fish have been reared in the laboratory using simple methods developed by home aquarists. For laboratories with high demand for breeding and generation turn-over, however, there has been a shift away from this approach towards one that leverages techniques, tools, and feeds from commercial aquaculture to help accelerate growth rates and decrease generation times. While these advances have improved efficiency, the effects of feeding zebrafish diets that are designed to grow disparately related cold-water fish species to market size quickly are not well-understood. To explore the impacts that intensive feeding protocols may have on this species, groups of zebrafish larvae from two different wild-type lines were stocked into treatment tanks at a standard density of 10 fish per liter and were administered either a "high" or "low" food diet for a maximum of 63 days. During their growth phase, the "high" food diet group received at least 8x more rotifers and at least 2x more Artemia than the "low" food diet group. Growth, survival, and reproductive performance (fecundity and viability) were measured in these fish and in their offspring. We found that fish that were fed more grew more rapidly and were able to reproduce earlier than fish that were fed less, but they were also more likely to produce higher proportions of non-viable embryos., Competing Interests: L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, Scholar Rock, and Branch Biosciences. He is a consultant for Celularity and Cellarity. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2022 Adatto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cellular barcodes are distinct DNA sequences that enable one to track specific cells across time or space. Recent advances in our ability to detect natural or synthetic cellular barcodes, paired with single-cell readouts of cell state, have markedly increased our knowledge of clonal dynamics and genealogies of the cells that compose a variety of tissues and organs. These advances hold promise to redefine our view of human disease. Here, we provide an overview of cellular barcoding approaches, discuss applications to gain new insights into disease mechanisms, and provide an outlook on future applications. We discuss unanticipated insights gained through barcoding in studies of cancer and blood cell production and describe how barcoding can be applied to a growing array of medical fields, particularly with the increasing recognition of clonal contributions in human diseases.

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

The zebrafish has proven to be a valuable model organism for studying hematopoiesis, but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single-cell RNA sequencing on ∼70,000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus, elucidated B- and T-cell developmental trajectories, and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic-like cell populations and provided evidence in support of the existence of a pre-B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies., (© 2022 Rubin et al.)

Spitz neoplasms are a diverse group of molecularly and histologically defined melanocytic tumors with varying biologic potentials. The precise classification of Spitz neoplasms can be challenging. Recent studies have revealed recurrent fusions involving multiple kinases in a large proportion of Spitz tumors. In this study, we generated a transgenic zebrafish model of Spitz melanoma using a previously identified ZCCHC8-ROS1 fusion gene. Animals developed grossly apparent melanocytic proliferations as early as 3 weeks of age and overt melanoma as early as 5 weeks. By 7 weeks, ZCCHC8-ROS1 induced a histologic spectrum of neoplasms ranging from hyperpigmented patches to melanoma. Given the swift onset of these tumors during development, we extended this approach into adult fish using a recently described electroporation technique. Tissue-specific expression of ZCCHC8-ROS1 in adults led to melanocyte expansion without overt progression to melanoma. Subsequent electroporation with tissue-specific CRISPR, targeting only tp53 was sufficient to induce transformation to melanoma. Our model exhibits the use of sequential mutagenesis in the adult zebrafish, and demonstrates that ZCCHC8-ROS1 induces a spectrum of melanocytic lesions that closely mimics human Spitz neoplasms., Competing Interests: Competing interests L.I.Z. is a founder and stockholder of Fate Therapeutics, Inc., Scholar Rock, Camp4 Therapeutics, Inc. and Amagma Therapeutics, Inc., (© 2022. Published by The Company of Biologists Ltd.)

Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo pharmacological perturbation of niche signals interferon, granulocyte colony-stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. External signals induced rapid transitions between HSC states but transcriptional response varied both between external stimulants and within the HSC population for a given perturbation. In contrast to LSK progenitors, HSCs were characterized by a greater link between molecular signatures at baseline and in response to external stressors. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq suggested some HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and LSK progenitor-specific chromatin and transcriptional features that represent determinants of signal receptiveness and regenerative potential during stress hematopoiesis., Competing Interests: EF, AS, MM, ER, SY, YZ, JG, NB, NB, FC No competing interests declared, DS is a director and equity holder of Agios Pharmaceuticals, Magenta Therapeutics, Editas Medicines, ClearCreekBio, and Life-VaultBio; a founder of Fate Therapeutics and Magenta Therapeutics; and a consultant to FOG Pharma and VCanBio, LZ is founder and stockholder of Fate, Inc, Scholar Rock, Camp4 therapeutics and a scientific advisor for Stemgent, (© 2021, Fast et al.)

Clonal hematopoiesis results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. We developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in clonal hematopoiesis–associated genes such as asxl1 promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1 , abrogated the ability of asxl1- mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny.