Development of an Undergraduate Cell Biology Laboratory to Assess Pigmentation and Cell Size in a Zebrafish Model of Uveal Melanoma.

This study outlines a 2-week laboratory module for an authentic cell biology undergraduate research experience that uses zebrafish (Danio rerio), a popular model organism for research. Previous research has indicated that course-based undergraduate research experiences such as this one increase student confidence, active learning, and retention. During this research experience, students investigate variations in pigmentation in the caudal fins of wild type (WT) and transgenic fish [Tg(mitfa:GNAQQ209L)]. The transgenic fish express a hyperactive Gα protein, GNAQQ209L, under the melanocyte-specific mitfa promoter, offering insights into uveal melanoma, a common eye cancer. Students specifically analyze the black pigmented cells, melanophores, within the caudal fin. We determined that the transgenic zebrafish have increased pigmentation in their caudal fins, but smaller melanophores. These results suggest there are more melanophores in the Tg(mitfa:GNAQQ209L) fish compared to the WT. Future undergraduate research could investigate these cellular differences. This research experience imparts microscopy and image analysis skills and instills the ability to grapple with large datasets, statistical tests, and data interpretation in alignment with biology education principles. Post-laboratory surveys reveal students attain confidence in the above skills and in handling animals, along with a deeper appreciation for model organism research and its relevance to cancer cell biology.

Genetic interference with HvNotch provides new insights into the role of the Notch-signalling pathway for developmental pattern formation in Hydra.

The Notch-signalling pathway plays an important role in pattern formation in Hydra. Using pharmacological Notch inhibitors (DAPT and SAHM1), it has been demonstrated that HvNotch is required for head regeneration and tentacle patterning in Hydra. HvNotch is also involved in establishing the parent-bud boundary and instructing buds to develop feet and detach from the parent. To further investigate the functions of HvNotch, we successfully constructed NICD (HvNotch intracellular domain)-overexpressing and HvNotch-knockdown transgenic Hydra strains. NICD-overexpressing transgenic Hydra showed a pronounced inhibition on the expression of predicted HvNotch-target genes, suggesting a dominant negative effect of ectopic NICD. This resulted in a "Y-shaped" phenotype, which arises from the parent-bud boundary defect seen in polyps treated with DAPT. Additionally, "multiple heads", "two-headed" and "ectopic tentacles" phenotypes were observed. The HvNotch-knockdown transgenic Hydra with reduced expression of HvNotch exhibited similar, but not identical phenotypes, with the addition of a "two feet" phenotype. Furthermore, we observed regeneration defects in both, overexpression and knockdown strains. We integrated these findings into a mathematical model based on long-range gradients of signalling molecules underlying sharply defined positions of HvNotch-signalling cells at the Hydra tentacle and bud boundaries.

Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges.

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.

Deciphering the genetic interactions between Pou4f3, Gfi1, and Rbm24 in maintaining mouse cochlear hair cell survival.

Mammals harbor a limited number of sound-receptor hair cells (HCs) that cannot be regenerated after damage. Thus, investigating the underlying molecular mechanisms that maintain HC survival is crucial for preventing hearing impairment. Intriguingly, Pou4f3-/- or Gfi1-/- HCs form initially but then rapidly degenerate, whereas Rbm24-/- HCs degenerate considerably later. However, the transcriptional cascades involving Pou4f3, Gfi1, and Rbm24 remain undescribed. Here, we demonstrate that Rbm24 expression is completely repressed in Pou4f3-/- HCs but unaltered in Gfi1-/- HCs, and further that the expression of both POU4F3 and GFI1 is intact in Rbm24-/- HCs. Moreover, by using in vivo mouse transgenic reporter assays, we identify three Rbm24 enhancers to which POU4F3 binds. Lastly, through in vivo genetic testing of whether Rbm24 restoration alleviates the degeneration of Pou4f3-/- HCs, we show that ectopic Rbm24 alone cannot prevent Pou4f3-/- HCs from degenerating. Collectively, our findings provide new molecular and genetic insights into how HC survival is regulated.

Functional identification of AeHMGR gene involved in regulation of saponin biosynthesis in Aralia elata.

Aralia elata (Miq.) Seem, a significant tree species in the Araliaceae family, has medicinal and edible properties. Saponins are the primary active components of A. elata. The 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR) is the initial rate-limiting enzyme of the major metabolic pathway of saponins in A. elata. In this study, the AeHMGR gene was identified through screening of transcriptome data. Through the qRT-PCR analysis, it was determined that the expression level of AeHMGR gene is highest in the somatic embryo and stem of A. elata. Heterologous transformation in tobacco revealed that ectopic expression of the AeHMGR gene leads to a significant reduction in the expression levels of the NtSS, NtFPS, and NtSE genes in transgenic tobacco lines, with a minimum expression level of 0.24 times that of the wild type. In the overexpressed callus lines of A. elata, the expression levels of the AeFPS, AeSE, AeSS, and Aeß-AS genes were also significantly lower compared to the wild type, with a minimum expression level of approximately 0.3 times that of the wild type. Interestingly, the overexpression of the AeHMGR gene in A. elata somatic embryos led to a substantial decrease in the expression levels of AeFPS and AeSS, while the expression levels of AeSE and Aeß-AS increased. Among the transgenic somatic embryo strain lines, line 7 exhibited the highest expression levels of AeSE and Aeß-AS, with fold increases of 11.51 and 9.38, respectively, compared with that of the wild-type. Additionally, a high-performance liquid chromatography method was established to detect five individual saponins in transgenic A. elata. The total saponin content in line 7 somatic embryos was 1.14 times higher than that of wild-type materials, but only 0.30 times that of wild-type cultivated leaves. Moreover, the content of oleanolic acid saponin in line 7 was 1.35 times higher than that of wild-type cultivated leaves. These indicate that HMGR can affect triterpene biosynthesis.

TCR-transgenic T cells and YB-1-based oncolytic virotherapy improve survival in a preclinical Ewing sarcoma xenograft mouse model.

Background: Ewing sarcoma (EwS) is an aggressive and highly metastatic bone and soft tissue tumor in pediatric patients and young adults. Cure rates are low when patients present with metastatic or relapsed disease. Therefore, innovative therapy approaches are urgently needed. Cellular- and oncolytic virus-based immunotherapies are on the rise for solid cancers. Methods: Here, we assess the combination of EwS tumor-associated antigen CHM1319-specific TCR-transgenic CD8+ T cells and the YB-1-driven (i.e. E1A13S-deleted) oncolytic adenovirus XVir-N-31 in vitro and in a xenograft mouse model for antitumor activity and immunostimulatory properties. Results: In vitro both approaches specifically kill EwS cell lines in a synergistic manner over controls. This effect was confirmed in vivo, with increased survival using the combination therapy. Further in vitro analyses of immunogenic cell death and antigen presentation confirmed immunostimulatory properties of virus-infected EwS tumor cells. As dendritic cell maturation was also increased by XVir-N-31, we observed superior proliferation of CHM1319-specific TCR-transgenic CD8+ T cells only in virus-tested conditions, emphasizing the superior immune-activating potential of XVir-N-31. Conclusion: Our data prove synergistic antitumor effects in vitro and superior tumor control in a preclinical xenograft setting. Combination strategies of EwS-redirected T cells and YB-1-driven virotherapy are a highly promising immunotherapeutic approach for EwS and warrant further evaluation in a clinical setting.

Genetically Encoded Reporters to Monitor Hypoxia.

Hypoxia resulting from an imbalance of oxygen availability and consumption defines a metabolic cellular state with a profound impact on developmental processes, tissue maintenance, and the development of pathologies. Fluorescence imaging using genetically encoded reporters enables hypoxia and oxygen imaging with cellular resolution. Thereby unrestricted visualization of hypoxic cells and regions essentially relies on the availability of oxygen-independent fluorescent proteins like UnaG, isolated from the Japanese freshwater eel. Here, we describe the application of recently developed members of a UnaG-based hypoxia reporter family to visualize oxygenation patterns by in vitro live-cell imaging and during the ex vivo analysis of intracranial xenografted tumors. Thus, the generation of stably transfected transgenic tumor cell lines, the in vitro calibration of the genetically encoded sensors, the surgical procedures for orthotopic xenografting of tumors in mice, and workflows for the respective sample preparation and microscopy are outlined.

Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production.

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.

Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.

Ribose-cysteine and levodopa abrogate Parkinsonism via the regulation of neurochemical and redox activities in alpha-synuclein transgenic Drosophila melanogaster models.

Parkinson's disease (PD), the most prevalent type of parkinsonism, is a progressive neurodegenerative condition marked by several non-motor and motor symptoms. PD is thought to have a complex aetiology that includes a combination of age, genetic predisposition, and environmental factors. Increased expression of α-synuclein (α-Syn) protein is central to the evolvement of neuropathology in this devastating disorder, but the potential of ribose-cysteine and levodopa in abating pathophysiologic changes in PD model is unknown. Crosses were set up between flies conditionally expressing a pathological variant of human α-Syn (UAS-α-Syn) and those expressing GAL4 in neurons (elav-GAL4) to generate offspring referred to as PD flies. Flies were randomly assigned to five groups (n = 40) from the total population of flies, with each group having five replicates. Groups of PD flies were treated with either 500 mg/kg ribose-cysteine diet, 250 mg/kg levodopa diet, or a combination of the two compounds for 21 days, whereas the control group (w1118) and the PD group were exposed to a diet without ribose-cysteine or levodopa. In addition to various biochemical and neurochemical assays, longevity, larval motility, and gravitaxis assays were carried out. Locomotive capability, lifespan, fecundity, antioxidant state, and neurotransmitter systems were all significantly (p < 0.05) compromised by overexpression of α-Syn. However, flies treated both ribose cysteine and levodopa showed an overall marked improvement in motor functions, lifespan, fecundity, antioxidant status, and neurotransmitter system functions. In conclusion, ribose-cysteine and levodopa, both singly and in combination, potentiated a therapeutic effect on alpha-synuclein transgenic Drosophila melanogaster models of Parkinsonism.

Sex differences in the tumor promoting effects of tobacco smoke in a cRaf transgenic lung cancer disease model.

Tobacco smoke (TS) is the leading cause for lung cancer (LC), and female smokers are at a greater risk for LC. Yet, the underlying causes are unknown. We performed whole genome scans in TS exposed wild type and histologically characterized tumor lesions of cRaf transgenic mice. We constructed miRNA-gene and transcription factor-miRNA/gene regulatory networks and determined sex-specific gene regulations by evaluating hormone receptor activities. We validated the findings from TS exposed cRaf mice in a large cohort of smoking and never-smoking LC patients. When compared to males, TS prompted a sevenfold increase in tumor multiplicity in cRaf females. Genome-wide scans of tumor lesions identified 161 and 53 genes and miRNAs, which code for EGFR/MAPK signaling, cell proliferation, oncomirs and oncogenes, and 50% of DEGs code for immune response and tumor evasion. Outstandingly, in transgenic males, TS elicited upregulation of 20 tumor suppressors, some of which are the targets of the androgen and estrogen receptor. Conversely, in females, 18 tumor suppressors were downregulated, and five were specifically repressed by the estrogen receptor. We found TS to perturb the circadian clock in a sex-specific manner and identified a female-specific regulatory loop that consisted of the estrogen receptor, miR-22-3p and circadian genes to support LC growth. Finally, we confirmed sex-dependent tumor promoting effects of TS in a large cohort of LC patients. Our study highlights the sex-dependent genomic responses to TS and the interplay of circadian clock genes and hormone receptors in the regulation of oncogenes and oncomirs in LC growth.

Benefits and opportunities of the transgenic Oncopig cancer model.

Cancer remains a leading cause of morbidity and mortality, and a paradigm shift is needed to fundamentally revisit drug development efforts. Pigs share close similarities to humans and may serve as an alternative model. Recently, a transgenic 'Oncopig' line has been generated to induce solid tumors with organ specificity, opening the potential of Oncopigs as a platform for developing novel therapeutic regimens.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

In Vivo Production of dsRNA Using Bacteriophage ϕ6 in Pseudomonas syringae Cit7 Cells.

RNA interference (RNAi), also known as post-transcriptional gene silencing (PTGS), is one of the emerging genetic engineering techniques to effectively silence or inhibit the expression of target genes. This chapter describes a method for in vivo production of dsRNA in non-pathogenic Pseudomonas syringae strains using phage ϕ6 RNA-dependent RNA polymerase, extraction and purification of dsRNA from bacterial solution, and the use of dsRNA to induce silencing of green fluorescent protein (GFP) in transgenic Nicotiana benthamiana.

Moderate dietary restriction delays the onset of age-associated sarcopenia in Caenorhabditis elegans due to reduced myosin UNC-54 degradation.

Sarcopenia, a gradual decrease in skeletal muscle mass and strength, is a major component of frailty in the elderly, with age, (lack of) exercise and diet found to be the major risk factors. The nematode Caenorhabditis elegans is an important model of sarcopenia. Although many studies describe loss of muscle function in ageing C. elegans, surprisingly few report on the loss of muscle mass. Here, in order to quantify loss of muscle mass under various dietary restriction (DR) conditions, we used an internal GFP standard to determine levels of the major body wall muscle myosin (UNC-54) in transgenic unc-54::gfp worms over their lifespan. Myosin density linearly increased during the first week of adulthood and there was no significant effect of DR. In contrast, an exponential decrease in myosin density was seen during the second week of adulthood, with reduced rates of myosin loss for mild and medium DR compared to control. UNC-54 turnover rates, previously determined using pulse-labelling methods, correspond well with the t1/2 value found here for UNC-54-GFP using fluorescence (control t1/2 = 12.0 days), independently validating our approach. These data indicate that sarcopenia is delayed in worms under mild and medium DR due to a reduced rate of myosin UNC-54 degradation, thereby maintaining protein homeostasis.

Firefly Rats: Illuminating the Scientific Community in Transplantation Research.

Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.

IL-1ß promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells.

ABSTRACT: JAK 2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1ß (IL-1ß)-mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1ß-/- or IL-1R1-/- mice. To study the role of IL-1ß and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1ß in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1ß levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1ß levels, and displayed low GFP-chimerism resembling CHIP. Anti-IL-1ß antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1ß as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

Single-cell RNA sequencing of a new transgenic t(8;21) preleukemia mouse model reveals regulatory networks promoting leukemic transformation.

T(8;21)(q22;q22), which generates the AML1-ETO fusion oncoprotein, is a common chromosomal abnormality in acute myeloid leukemia (AML) patients. Despite having favorable prognosis, 40% of patients will relapse, highlighting the need for innovative models and application of the newest technologies to study t(8;21) leukemogenesis. Currently, available AML1-ETO mouse models have limited utility for studying the pre-leukemic stage because AML1-ETO produces mild hematopoietic phenotypes and no leukemic transformation. Conversely, overexpression of a truncated variant, AML1-ETO9a (AE9a), promotes fully penetrant leukemia and is too potent for studying pre-leukemic changes. To overcome these limitations, we devised a germline-transmitted Rosa26 locus AE9a knock-in mouse model that moderately overexpressed AE9a and developed leukemia with long latency and low penetrance. We observed pre-leukemic alterations in AE9a mice, including skewing of progenitors towards granulocyte/monocyte lineages and replating of stem and progenitor cells. Next, we performed single-cell RNA sequencing to identify specific cell populations that contribute to these pre-leukemic phenotypes. We discovered a subset of common myeloid progenitors that have heightened granulocyte/monocyte bias in AE9a mice. We also observed dysregulation of key hematopoietic transcription factor target gene networks, blocking cellular differentiation. Finally, we identified Sox4 activation as a potential contributor to stem cell self-renewal during the pre-leukemic stage.