T cells expressing the γδ T cell receptor (TCR) develop in a stepwise process initiating at the αβ/γδ T cell lineage choice followed by maturation and acquisition of effector functions, including the ability to produce interleukin-17 (IL-17) as γδT17 cells. Previous studies linked TCR signal strength and T cell fate choices to the transcriptional regulator HEB (encoded by Tcf12) and its antagonist, Id3, but how these factors regulate different stages of γδ T cell development has not been determined. We found that immature fetal γδTCR+ cells from conditional Tcf12 knockout (HEB cKO) mice were defective in activating the γδT17 program at an early stage, whereas Id3 deficient (Id3-KO) mice displayed a partial block in γδT17 maturation and an inability to produce IL-17. We also found that HEB cKO mice failed to upregulate Id3 during γδT17 development, whereas HEB overexpression elevated the levels of Id3 in collaboration with TCR signaling. Moreover, Egr2 and HEB were bound to several of the same regulatory sites on the Id3 gene locus in the context of early T cell development. Therefore, our findings reveal an interlinked sequence of events during which HEB and TCR signaling synergize to upregulate Id3, which enables maturation and acquisition of the γδT17 effector program.

The thymus is a primary lymphoid organ in which diverse and self-tolerant T cells are produced from bone marrow-derived hematopoietic progenitors. Progressive, age-associated thymic involution reduces T-cell output and impairs adaptive immunity; however, the molecular mechanisms underlying this process remain elusive. Here, we report that the conditional deletion of the RNA-binding proteins Zfp36l1 and Zfp36l2 in thymic epithelial cells (TECs) leads to a pronounced reduction in the number of TECs during the embryonic stage and early neonatal stage, despite a largely preserved thymus size. Postnatally, these mice exhibit excessive medullary TEC (mTEC) expansion, elevated intrathymic proinflammatory cytokine production, FOXN1 downregulation, and premature thymic involution. These findings reveal a protective role for Zfp36 Tristetraprolin (TTP) family proteins in regulating cytokine levels within the thymic microenvironment and preventing premature thymic involution. Moreover, our results suggest a previously unappreciated connection between central tolerance induction and the onset of age-associated thymic involution.

Thymic macrophages (TMs) are critical for maintaining thymus homeostasis, yet their heterogeneity and specific functions in T cell development remain unclear. Through single-cell RNA sequencing and transgenic reporter mice, we revealed two novel TM subsets characterized by distinct surface markers and transcriptional programs. TIMD4⁺ VCAM1⁺ CX3CR1⁻ TMs were enriched for phagocytic and apoptotic-cell–clearance pathways, whereas TIMD4⁻ VCAM1⁺ CX3CR1⁺ TMs exhibited elevated expression of antigen-presentation machinery and interferon-response genes. Pseudotime trajectory analysis indicated a potential lineage progression from TIMD4⁻ VCAM1⁺ CX3CR1⁺ precursors toward TIMD4⁺ VCAM1⁺ CX3CR1⁻ effectors. Functional interrogation using fetal thymic organ cultures and the MaFIA depletion mouse model demonstrated that acute loss of TMs severely impaired thymocyte development, particularly at the pre-T cell receptor (TCR) β-selection checkpoint. TM ablation led to marked reductions in post-β-selection thymocyte subsets, implicating TMs in both survival and differentiation signals required for early T cell development. Mechanistically, TM depletion disrupted pre-TCR signaling and hindered transition through β-selection, likely due to absence of both apoptotic-cell clearance and differentiation cues provided by specialized TM subsets. These findings define two distinct TM populations and establish their role in orchestrating thymocyte maturation. By elucidating TM subset specialization and their dynamic contributions to the β-selection process, our findings advance our understanding of thymic microenvironment interactions and highlights macrophage heterogeneity as a key regulator of T cell development.

Hematopoietic stem and progenitor cells (HSPCs) differentiate into diverse blood cell lineages through intricate genetic and epigenetic decision processes. How these decision points are regulated has not been fully elucidated. We aimed to identify the factors influencing human HSPC differentiation into T lymphocytes using a single-cell RNA sequencing-based lineage tracing approach. Our approach revealed that a bifurcation between the mast cell and lympho-myeloid lineage trajectories occurs early, with lymphoid and myeloid clones remaining interconnected until later time points, when T cell lineage commitment is established. We identified a new mechanism that primes certain HSPCs to become T cells, even though all cells express NOTCH receptors and are exposed to Notch ligands. GXYLT2, a xylosyltransferase that regulates NOTCH activation post-translationally, was differentially expressed in HSPCs with a stronger T cell clonal bias. Considering this cell-intrinsic, heritable priming state, we investigated the factors influencing lineage priming before Notch ligand exposure. We discovered that class 1 histone deacetylases (HDACs) favor lymphoid outcomes, with HDAC inhibition promoting mast cell outcomes and activation enhancing T-lymphoid differentiation. Additionally, we identified binding regions for IRF1 and GATA3, which are early predictors of human lymphoid outcomes within the GXYLT2 promoter region. Further analysis of the GXYLT2 loci revealed a retrotransposon element that acts as a regulator of the loci, appearing to be evolutionarily divergent but conserved among primates. Removing this element increased T cell bias in our cultures. Our findings revealed a previously unrecognized role for GXYLT2 and its loci in influencing early decisions toward human T cell development outcomes.

Hematopoiesis originates from hematopoietic stem and progenitor cells (HSPCs), which can self-renew and differentiate to create diverse, functional blood cells. The process through which the HSPCs give rise to differentiated cells involves intricate genetic and epigenetic decision-making. Our group previously developed an in vitro culture system in which CD34+ cells from various sources faithfully produce T-lineage cells (Shukla et al., 2017). Using this system, we sought to understand the signaling intricacies involved in HSPC's expedition to become T cells. To achieve this, we performed a lineage tracing experiment by labeling umbilical cord blood-derived CD34+ cells with lentiviral barcodes. The HSPCs were then differentiated into pro-T cells and longitudinally sampled (days 0, 3, 6, and 9) for single-cell RNAseq (scRNAseq). Our analysis revealed three distinct trajectories, one leading to the successful differentiation of T-lineage cells and the others culminating in myeloid and mast cell lineage outcomes. When we examined the lineage tracing data, we found that the divergence to the mast cell lineage occurs early in the differentiation process, with GATA1 expression driving mast cell fate. The lympho-myeloid trajectories are interwind until later, even the strongly lymphoid-biased clones showed lymphoid-myeloid bipotential, simultaneously expressing both myeloid transcription factors (SPI1, IRF8), lymphoid transcription factor (BCL11B) and multipotent factors (RUNX1 and RUNX3) until complete transition, underscoring the nuances in fate decision. We identified approximately 1600 genes that significantly drove lymphoid potential. Among the major transcription factors, GATA3 expression is the early predictor of T-lineage clonal outcome (at day 0), a key finding that significantly advances our understanding of T-cell differentiation. NOTCH responsiveness was identified as a later predictor (on day 3), with NOTCH target genes DTX1 and NRARP response predicting successful T-lymphoid outcomes. From the list of significant genes, we created a gene signature that can predict lymphoid outcomes using this data. We then collected samples at day 1 (CD34+ CD38-), day 6 (CD7+), and day 14 (CD7+ CD5+, CD7+CD1a+) of differentiation and performed a scRNA-seq and scATAC-seq on sorted cells simultaneously. We applied the lymphoid predictive gene signature to the dataset; we could see the gene signature expressing as early as day 1, even before the initiation of BCL11B, with about one-third of CD34+ CD38- HSPCs in culture expressing the gene signature. Interestingly, the expression of this gene signature progressively increased until day six and slightly reduced at day 14 after the cells acquired CD5, suggesting that the gene set may be more critical in the initial time points. Given that the gene set, when probed for the gene-disease association, had a strong correlation to T acute lymphoid leukemia (T-ALL) and the requirement of these genes at initial time points, we think a transient expression of some of these factors could help us fate engineer HSCs to produce lymphoid outcomes. We are validating the significant genes whose transient expression might lead to T-cell fate engineering without predisposing these cells to T-ALL.

Two major developmental steps are necessary for the generation of innate IL-17 producing γδ T cells. First, T cell precursors in the fetal thymus commit to either the αβ or the γδ T cell lineage. This is followed by a second stage of differentiation into subsets that can produce either IL-17 (γδT17) or IFNγ (γδT1) cells. Both developmental events are dependent on TCR signal strength, but how this is interpreted at the molecular level has been unclear. Id3, which antagonizes the activity of HEB transcription factors, is upregulated in proportion to TCR signal strength, providing an important link between TCR signaling and gene expression. We previously found that γδ T cells in mice lacking HEB were impaired in IL-17 production. To better understand the molecular basis of this defect, we conducted single cell RNA-sequencing on fetal thymic γδ T cells. In WT cells, the ratio of HEB to Id3 was highest in populations undergoing γδ T cell commitment, whereas later populations were dominated by Id3. Comparing scRNA-seq datasets from WT and HEB-deficient γδ T cells revealed a profound shift in TCRγ and TCRδ chain expression. This was accompanied by a decrease in key regulators of early γδ T cell development. Id3 expression was also severely decreased. Accordingly, Id3-deficient mice were impaired in their ability to generate γδT17 cells. However, γδ T cells from Id3-deficient mice showed a decrease in second stage γδT17 regulators, rather than the first stage regulators defective in HEB-deficient mice. Therefore, distinct ratios of HEB and Id3 are required during the γδ T cell commitment, and HEB is instrumental in inducing Id3 to enable functional programming of γδT17 cells in the fetal thymus. Supported by grants from CIHR (201610PJT), NSERC (RGPIN-2020-05596) and NIH (1P01AI102853-06)

Zebrafish offer an excellent tool for studying the vertebrate hematopoietic system thanks to a highly conserved and rapidly developing hematopoietic program, genetic amenability, optical transparency, and experimental accessibility. Zebrafish studies have contributed to our understanding of hematopoiesis, a complex process regulated by signaling cues, inflammation being crucial among them. Hematopoietic stem cells (HSCs) are multipotent cells producing all the functional blood cells, including immune cells. HSCs respond to inflammation during infection and malignancy by proliferating and producing the blood cells in demand for a specific scenario. We first focus on how inflammation plays a crucial part in steady-state HSC development and describe the critical role of the inflammasome complex in regulating HSC expansion and balanced lineage production. Next, we review zebrafish studies of inflammatory innate immune mechanisms focusing on interferon signaling and the downstream JAK-STAT pathway. We also highlight insights gained from zebrafish models harbouring genetic perturbations in the role of inflammation in hematopoietic disorders such as bone marrow failure, myelodysplastic syndrome, and myeloid leukemia. Indeed, inflammation has been recently identified as a potential driver of clonal hematopoiesis and leukemogenesis, where cells acquire somatic mutations that provide a proliferative advantage in the presence of inflammation. Important insights in this area come from mutant zebrafish studies showing that hematopoietic differentiation can be compromised by epigenetic dysregulation and the aberrant induction of signaling pathways.

KIT, a type III tyrosine kinase receptor, plays a crucial role in haematopoietic development. The KIT receptor forms a dimer after ligand binding; this activates tyrosine kinase activity leading to downstream signal transduction. The D816V KIT mutation is extensively implicated in haematological malignancies, including mastocytosis and leukaemia. KIT D816V is constitutively active, but the molecular nuances that lead to constitutive tyrosine kinase activity are unclear. For the first time, we present experimental evidence that the KIT D816V mutant does not dimerize like KIT wild type. We further show evidence of decreased stabilization of the tyrosine kinase domain in the KIT D816V mutant, a phenomenon that might contribute to its constitutive activity. Since the mechanism of KIT D816V activation varies from that of the wild type, we explored downstream signal transduction events and found that even though KIT D816V targets similar signalling moieties, the signalling is amplified in the mutant compared to stem cell factor-activated wild type receptor. Uniquely, KIT D816V induces infection-related pathways and the spliceosome pathway, providing alternate options for selective as well as combinatorial therapeutic targeting.

TET2 loss-of-function mutations are recurrent events in a wide range of hematological malignancies and a physiologic occurrence in blood cells of healthy older adults. It is currently unknown what determines if a person harboring a somatic TET2 mutation will progress to myelodysplastic syndrome or acute myeloid leukemia. Here we develop a zebrafish tet2 mutant through which we show that tet2 loss leads to restricted hematopoietic differentiation combined with a modest upregulation of p53, which is also characteristic of many inherited bone marrow failure syndromes. Uniquely in the context of emergency hematopoiesis by external stimuli, such as infection or cytokine stimulation, lack of tet2 leads hematopoietic stem cells to undergo excessive proliferation, resulting in an accumulation of immature cells, which are poised to become leukemogenic following additional genetic/epigenetic perturbations. This same phenomenon observed in zebrafish extends to human hematopoietic stem cells, identifying TET2 as a critical relay switch in the context of stress hematopoiesis.

Background: Knock-in of precise point mutations into protein-coding genes is among the most important applications of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9. Precise introduction of amino acid changes is crucial to interrogate the function of specific protein residues and to create human disease models. Mutations of homologous protein residues in model animal species enable the studies of their consequences, leading to a better understanding of the disease in question. Objectives: Manual design of point mutation knock-ins is a time-consuming process consisting of many steps assisted by several computational tools. We have, therefore, designed CRISPR Knock-in Designer, which can perform the rapid and automatic design of point mutation knock-in DNA oligonucleotides upon provision of the mutation, a guide RNA, and identifier or sequence information. Method: We have used the shiny application framework in R for developing the website and several relevant R packages for the data processing and visualization steps. Automatic download of the relevant data occurs from the REST application programming interfaces provided by Ensembl. Results: The tool supports most experimentally established CRISPR types and has multiple options for the resulting oligonucleotides. We also provide allele-specific polymerase chain reaction-based and restriction enzymebased genotyping strategies in the program output. CRISPR Knock-in Designer adjusts to the genomic context of any target codon and designs knock-in strategies for two-exon straddling codons, which we explored in multiple species. CRISPR Knock-in Designer also provides input for two Prime Editing design tools to facilitate the introduction of a specific mutation sequence using this advanced technology. Conclusions: CRISPR Knock-in Designer provides an automatic way to design CRISPR/Cas-based amino acid substitution knock-in strategies including the genotyping strategies and generates inputs for Prime Editing design software.

Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lack specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first humanized zebrafish that express human hematopoietic-specific cytokines (GM-CSF, SCF, and SDF1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation that more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.

Cholesterol biosynthesis is intrinsically linked to the regulation of hematopoiesis

Human hematopoietic stem cells (HSCs) are characterized by their unique ability to self-renew and differentiate into multiple cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating the in vivo repopulation capacity of these cells and has been adapted to evaluate the leukemic potential of leukemia-initiating cells (LICs) in acute leukemia. In spite of the popularity of this approach, certain cellular subsets do not survive post xenotransplantation as they are dependent on human factors that may be missing in the murine microenvironment. We pioneered human leukemia xenografts in the zebrafish model (Corkery et al., 2011; Bentley et al., 2015) as a parallel strategy, taking advantage of the immuno-permissiveness and transparency of zebrafish larvae to provide unprecedented continuous observation of leukemia evolution. However, as in mice, the addition of human cytokines and growth factors may further improve engraftment and optimize the utility of the zebrafish as a preclinical transplant model for medium to high-throughput therapeutic screening by creating a microenvironment more comparable to that found in patients.

The hallmark of hematopoietic stem cells (HSCs) is their ability to self-renew and differentiate into multiple blood cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating functionality and has been adapted to study leukemia initiating cells (LICs). However, specific cellular subsets do not survive post-xenotransplantation, due to a dependence on factors that may be missing in a non-primate microenvironment.

Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia.

BACKGROUND: Plumbagin (5-hydroxy-2-methyl-1,4-napthoquinone) derived from Plumbago species is a potential anti-tumour agent. Plumbagin has been tested for anti-cancer activity in vitro and in vivo using mice model. AIM: To study the tumour suppressing efficacy of plumbagin using zebrafish model. MATERIALS AND METHODS: Human Non-small lung cancer cell line were cultured in vitro and transplanted in to zebrafish. The development of tumour was confirmed by performing histology. The tumour was then allowed to progress in vivo and the fishes were administered with plumbagin orally for three continuous days. The tumour suppression capacity was monitored subsequently using transcriptosome analysis. STATISTICAL METHODS: The pixel integrated density obtained was converted into relative gene expression using IBM SPSS. RESULTS: The administration of plumbagin had an ability to suppress tumour and the size of the tumour were relatively lesser when compared with the control sample; it has also increased p53 gene expression. CONCLUSION: The study helps to conclude that plumbagin is an effective anti-tumour agent against human cancer cells based on the study in vivo in zebrafish.

The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.