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B cell lymphoma 6 (BCL6) is a conserved multi-domain protein that functions principally as a transcriptional repressor. This protein regulates many pivotal aspects of immune cell development and function. BCL6 is critical for germinal center (GC) formation and the development of high-affinity antibodies, with key roles in the generation and function of GC B cells, follicular helper T (Tfh) cells, follicular regulatory T (Tfr) cells, and various immune memory cells. BCL6 also controls macrophage production and function as well as performing a myriad of additional roles outside of the immune system. Many of these regulatory functions are conserved throughout evolution. The BCL6 gene is also important in human oncology, particularly in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), but also extending to many in other cancers, including a unique role in resistance to a variety of therapies, which collectively make BCL6 inhibitors highly sought-after.

The suppressor of cytokine signaling (SOCS) family of proteins were named after their defining role as negative feedback regulators of signaling initiated by numerous cytokine receptors. However, multiple members of the SOCS family likely function outside of this paradigm, including SOCS4. Zebrafish possess two SOCS4 paralogues, with socs4a previously shown to participate in central nervous system development and function. This study examined the role of the other paralogue, socs4b , through expression analysis and functional investigations in vivo and in vitro. This revealed maternal deposition of socs4b mRNA, specific zygotic expression during late embryogenesis, including in the brain, eye and intestine, and broad adult expression that was highest in the brain. A mutant allele, socs4bΔ18 , was generated by genome editing, in which the start codon was deleted. Fish homozygous for this likely hypomorphic allele showed no overt developmental phenotypes. However, in vitro studies suggested the Socs4b protein may be able to regulate EGFR signaling.

All families deserve access to readily available, accurate, and relevant information to help them navigate the newborn screening system. Current practices, limited resources, and a siloed newborn screening system create numerous challenges for both providers and families to implement educational opportunities to engage families in ways that meet their needs with relevant and meaningful approaches. Engaging families in newborn screening, especially those from historically underserved communities, is necessary to increase knowledge and confidence which leads to overall improved outcomes for families. This article describes three strategies that the Navigate Newborn Screening Program developed, tested, and implemented in the United States, including online learning modules, a prenatal education pilot program, and social media awareness campaign, as well as the extent to which they were successful in reaching and educating families about newborn screening. Using quality improvement methods and evidence-driven approaches, each of these three strategies demonstrate promising practices for advancing awareness, knowledge, and self-efficacy for families navigating the newborn screening system-particularly families in medically underserved and underrepresented communities. A model for bidirectional engagement of families is outlined to support scaling and implementing promising educational efforts for both providers and families in the newborn screening system.

The Janus kinase (JAK) family is a small group of protein tyrosine kinases that represent a central component of intracellular signaling downstream from a myriad of cytokine receptors. The JAK3 family member performs a particularly important role in facilitating signal transduction for a key set of cytokine receptors that are essential for immune cell development and function. Mutations that impact JAK3 activity have been identified in a number of human diseases, including somatic gain-of-function (GOF) mutations associated with immune cell malignancies and germline loss-of-function (LOF) mutations associated with immunodeficiency. The structure, function and impacts of both GOF and LOF mutations of JAK3 are highly conserved, making animal models highly informative. This review details the biology of JAK3 and the impact of its perturbation in immune cell-related diseases, including relevant animal studies.

Myeloproliferative neoplasms (MPNs) are hematopoietic diseases characterized by the clonal expansion of single or multiple lineages of differentiated myeloid cells that accumulate in the blood and bone marrow. MPNs are grouped into distinct categories based on key clinical presentations and distinctive mutational hallmarks. These include chronic myeloid leukemia (CML), which is strongly associated with the signature BCR::ABL1 gene translocation, polycythemia vera (PV), essential thrombocythemia (ET), and primary (idiopathic) myelofibrosis (PMF), typically accompanied by molecular alterations in the JAK2 , MPL , or CALR genes. There are also rarer forms such as chronic neutrophilic leukemia (CNL), which involves mutations in the CSF3R gene. However, rather than focusing on the differences between these alternate disease categories, this review aims to present a unifying molecular etiology in which these overlapping diseases are best understood as disruptions of normal hematopoietic signaling: specifically, the chronic activation of signaling pathways, particularly involving signal transducer and activator of transcription (STAT) transcription factors, most notably STAT5B, leading to the sustained stimulation of myelopoiesis, which underpins the various disease sequalae.

Introduction: Signal transducer and activator of transcription (STAT) 3 is extensively involved in the development, homeostasis, and function of immune cells, with STAT3 disruption associated with human immune-related disorders. The roles ascribed to STAT3 have been assumed to be due to its canonical mode of action as an inducible transcription factor downstream of multiple cytokines, although alternative noncanonical functional modalities have also been identified. The relative involvement of each mode was further explored in relevant zebrafish models., Methods: Genome editing with CRISPR/Cas9 was used to generate mutants of the conserved zebrafish Stat3 protein: a loss of function knockout (KO) mutant and a mutant lacking C-terminal sequences including the transactivation domain (ΔTAD). Lines harboring these mutations were analyzed with respect to blood and immune cell development and function in comparison to wild-type zebrafish., Results: The Stat3 KO mutant showed perturbation of hematopoietic lineages throughout primitive and early definitive hematopoiesis. Neutrophil numbers did not increase in response to lipopolysaccharide (LPS) or granulocyte colony-stimulating factor (G-CSF) and their migration was significantly diminished, the latter correlating with abrogation of the Cxcl8b/Cxcr2 pathway, with macrophage responses perturbed. Intriguingly, many of these phenotypes were not shared by the Stat3 ΔTAD mutant. Indeed, only neutrophil and macrophage development were disrupted in these mutants with responsiveness to LPS and G-CSF maintained, and neutrophil migration actually increased., Conclusion: This study has identified roles for zebrafish Stat3 within hematopoietic stem cells impacting multiple lineages throughout primitive and early definitive hematopoiesis, myeloid cell responses to G-CSF and LPS and neutrophil migration. Many of these roles showed conservation, but notably several involved noncanonical modalities, providing additional insights for relevant diseases., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated canonically by numerous cytokines and other factors, with significant roles in immunity, immune diseases, and cancer. It has also been implicated in several human skeletal disorders, with loss-of-function (LOF) mutations associated with aberrant skeletal development. To gain further insights, two zebrafish STAT3 lines were investigated: a complete LOF knockout (KO) mutant and a partial LOF mutant with the transactivation domain truncated (ΔTAD). Consistent with other studies, the KO mutants were smaller, with reduced length in early embryos exacerbated by a decreased growth rate from 5 days postfertilization (dpf). They displayed skeletal deformities that approached 80% incidence by 30 dpf, with a significant reduction in early bone but not cartilage formation. Further analysis additionally identified considerable abrogation of caudal fin regeneration, concomitant with a paucity of infiltrating macrophages and neutrophils, which may be responsible for this. Most of these phenotypes were also observed in the ΔTAD mutants, indicating that loss of canonical STAT3 signaling was the likely cause. However, the impacts on early bone formation and regeneration were muted in the ΔTAD mutant, suggesting the potential involvement of noncanonical functions in these processes.

The signal transducer and activator of transcription (STAT) family of proteins has been demonstrated to perform pivotal roles downstream of a myriad of cytokines, particularly those that control immune cell production and function. This is highlighted by both gain-of-function (GOF) and loss-of-function (LOF) mutations being implicated in various diseases impacting cells of the immune system. These mutations are typically inherited, although somatic GOF mutations are commonly observed in certain immune cell malignancies. This review details the growing appreciation of STAT proteins as a key node linking immunodeficiency, autoimmunity and cancer.

The Cytokine-inducible Src homology 2 domain-containing (CISH) protein is a negative feedback regulator induced by cytokines that play key roles in immunity and erythropoiesis. Single nucleotide polymorphisms (SNPs) in the human CISH gene have been associated with increased susceptibility to severe malaria disease. To directly assess how CISH might influence outcomes in the BALB/c model of malaria anemia, CISH knockout ( Cish -/- ) mice on this background were infected with Plasmodium berghei and their hematopoietic responses, cytokine production and ability to succumb to severe malaria disease evaluated. Despite basal erythrocytic disruption, upon P. berghei infection, the Cish -/- mice were better able to maintain peripheral blood cell counts, hemoglobin levels and a steady-state pattern of erythroid differentiation compared to wild-type ( Cish +/+ ) mice. Ablation of CISH, however, did not influence the outcome of acute malaria infections in either the BALB/c model or the alternative C57BL/6 model of experimental cerebral malaria, with the kinetics of infection, parasite load, weight loss and cytokine responses being similar between Cish +/+ and Cish -/- mice, and both genotypes succumbed to experimental cerebral malaria within a comparable timeframe., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Lakkavaram, Maymand, Naser, Ward and de Koning-Ward.)

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

Background: Signal transducer and activator of transcription (STAT) proteins play key roles in development, growth, and homeostasis. These roles have principally been assigned to their "canonical" function as inducible transcriptional activators acting downstream of cytokines and other factors. However, variant "non-canonical" functions have also been identified. The potential in vivo role for non-canonical STAT functions was investigated in the zebrafish model., Methods: Two zebrafish Stat5.1 mutants were generated using CRISPR/Cas9 that should impact canonical functionality: one with a deleted transactivation domain (ΔTAD) and another with a disrupted tyrosine motif (ΔTM). Immune cell development, growth, and adiposity of these Stat5.1 mutants were assessed in comparison to a Stat5.1 knockout (KO) mutant in which both canonical and non-canonical functions were ablated., Results: Both the ΔTAD and ΔTM mutants showed significantly reduced embryonic T lymphopoiesis, similar to the KO mutant. Additionally, adult ΔTAD and ΔTM mutants displayed a decrease in T cell markers in the kidney, but not as severe as the KO, which also showed T cell disruption in the spleen. Severe growth deficiency and increased adiposity were observed in all mutants, but ΔTAD showed a more modest growth defect whereas ΔTM exhibited more profound impacts on both growth and adiposity, suggesting additional gain-of-function activity., Conclusions: These results indicate that canonical Stat5.1 plays a major role in T cell development and growth throughout the lifespan and non-canonical Stat5.1 functions also contribute to aspects of adult T lymphocyte development and growth, with alternate functions impacting growth and adiposity., Competing Interests: The authors declare no conflict of interest. Given his role as Guest Editor, ACW had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Amedeo Amedei., (© 2023 The Author(s). Published by IMR Press.)

Cytokine-inducible SH2 domain-containing protein (CISH) is a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators shown to play crucial roles in lymphoid cell development and function as well as appetite regulation. It has also been implicated in the control of signaling downstream of the receptors for the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in myeloid cells. To investigate the physiological role of CISH in myelopoiesis, mice deficient in CISH were analyzed basally and in response to administration of these cytokines. CISH knockout (KO) mice possessed basally elevated neutrophils in the blood, bone marrow, and spleen compared to wild-type (WT) mice. During GM-CSF-induced myelopoiesis, the frequency of neutrophils, myeloid dendritic cells (DCs), and CFU-M in the bone marrow was higher in the KO, as were the neutrophils and CFU-G in the spleen. In contrast, no differences were observed between KO and WT mice during G-CSF-induced myelopoiesis apart from an elevated frequency of CFU-G and CFU-M in the spleen. This work has identified a role for CISH in the negative regulation of granulopoiesis, including that mediated by GM-CSF.

Tetracycline antibiotics are used extensively in veterinary medicine, but the majority of the administrated dose is eliminated unmodified from the animal through various excretion routes including urine, faeces and milk. In dairy animals, limits on residues secreted in milk are strictly controlled by legislation. Tetracyclines (TCs) have metal chelation properties and form strong complexes with iron ions under acidic conditions. In this study, we exploit this property as a strategy for low cost, rapid electrochemical detection of TC residues. TC-Fe(III) complexes in a ratio of 2:1 were created in acidic conditions (pH 2.0) and electrochemically measured on plasma-treated gold electrodes modified with electrodeposited gold nanostructures. DPV measurements showed a reduction peak for the TC-Fe(III) complex that was observed at 50 mV (vs. Ag/AgCl QRE). The limit of detection in buffer media was calculated to be 345 nM and was responsive to increasing TC concentrations up to 2 mM, added to 1 mM FeCl3. Whole milk samples were processed to remove proteins and then spiked with tetracycline and Fe(III) to explore the specificity and sensitivity in a complex matrix with minimal sample preparation, under these conditions the LoD was 931 nM. These results demonstrate a route towards an easy-to-use sensor system for identification of TC in milk samples taking advantage of the metal chelating properties of this antibiotic class., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Raykova 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.)

Background: Family and domestic violence, encompassing diverse behaviours including physical, sexual, emotional and financial abuse, is endemic worldwide and has multiple adverse health and social consequences. Principal drivers include traditional gender values that disempower women. Changing these is a key prevention strategy. In Australia, high-quality national surveys provide data on public perspectives concerning family and domestic violence but may not capture community-level diversity. As part of a project for primary prevention family and domestic violence in outer regional Australia, our aims were to develop and administer a questionnaire-based survey suitable for the local community encompassing knowledge about, attitudes towards, and personal experiences of family and domestic violence, to describe and to investigate the theoretical (factor) structure and local socio-demographic predictors of responses, and to determine the extent to which the survey findings are locally distinctive., Methods: The online community survey for local residents (≥15 years), comprised items on respondents' sociodemographic characteristics plus questions abridged from pre-existing national instruments on knowledge about, attitudes towards, and personal experiences of family and domestic violence. Responses were rake-weighted to correct census-ascertained sample imbalance and investigated using exploratory factor analysis, with sociodemographic predictors determined using multiple linear regression and dominance analysis., Results: Among 914 respondents, males (27.0%), those from age-group extremes, and less-educated persons were underrepresented. Familiarity with diverse family and domestic violence behaviours was high among all subgroups. Poorer knowledge of the FDV behaviour continuum and attitudes supporting traditional gender roles and FDV were disproportionately evident among males, older respondents and those with lower education levels. Both the factor structure of extracted composite measures reflecting community perspectives and sociodemographic predictors of responses generally aligned with patterns evident in national data., Conclusions: Local reinforcement of existing nationwide findings on community understanding of and attitudes towards family and domestic violence provides salience for targeted interventions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Woods 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.)

Signal transducer and activator of transcription (STAT) proteins act downstream of cytokine receptors to facilitate changes in gene expression that impact a range of developmental and homeostatic processes. Patients harbouring loss-of-function (LOF) STAT5B mutations exhibit postnatal growth failure due to lack of responsiveness to growth hormone as well as immune perturbation, a disorder called growth hormone insensitivity syndrome with immune dysregulation 1 (GHISID1). This study aimed to generate a zebrafish model of this disease by targeting the stat5.1 gene using CRISPR/Cas9 and characterising the effects on growth and immunity. The zebrafish Stat5.1 mutants were smaller, but exhibited increased adiposity, with concomitant dysregulation of growth and lipid metabolism genes. The mutants also displayed impaired lymphopoiesis with reduced T cells throughout the lifespan, along with broader disruption of the lymphoid compartment in adulthood, including evidence of T cell activation. Collectively, these findings confirm that zebrafish Stat5.1 mutants mimic the clinical impacts of human STAT5B LOF mutations, establishing them as a model of GHISID1., (© 2023. The Author(s).)

Primary immunodeficiency (PID) disorders, also commonly referred to as inborn errors of immunity, are a heterogenous group of human genetic diseases characterized by defects in immune cell development and/or function. Since these disorders are generally uncommon and occur on a variable background profile of potential genetic and environmental modifiers, animal models are critical to provide mechanistic insights as well as to create platforms to underpin therapeutic development. This review aims to review the relevance of zebrafish as an alternative genetic model for PIDs. It provides an overview of the conservation of the zebrafish immune system and details specific examples of zebrafish models for a multitude of specific human PIDs across a range of distinct categories, including severe combined immunodeficiency (SCID), combined immunodeficiency (CID), multi-system immunodeficiency, autoinflammatory disorders, neutropenia and defects in leucocyte mobility and respiratory burst. It also describes some of the diverse applications of these models, particularly in the fields of microbiology, immunology, regenerative biology and oncology.

Introduction: Suppressor of cytokine signaling 3 (SOCS3) is a critical component of the negative feedback regulation that controls signaling by cytokines and other factors thereby ensuring that important processes such as hematopoiesis and inflammation occur at appropriate levels., Methods: To gain further insights into SOCS3 function, the zebrafish socs3b gene was investigated through analysis of a knockout line generated using CRISPR/Cas9-mediated genome editing., Results: Zebrafish socs3b knockout embryos displayed elevated numbers of neutrophils during primitive and definitive hematopoiesis but macrophage numbers were not altered. However, the absence of socs3b reduced neutrophil functionality but enhanced macrophage responses. Adult socs3b knockout zebrafish displayed reduced survival that correlated with an eye pathology involving extensive infiltration of neutrophils and macrophages along with immune cell dysregulation in other tissues., Discussion: These findings identify a conserved role for Socs3b in the regulation of neutrophil production and macrophage activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sobah, Scott, Laird, Koole, Liongue and Ward.)

Introduction: The granulocyte colony-stimulating factor receptor (G-CSFR), encoded by the CSF3R gene, is involved in the production and function of neutrophilic granulocytes. Somatic mutations in CSF3R leading to truncated G-CSFR forms are observed in acute myeloid leukemia (AML), particularly those subsequent to severe chronic neutropenia (SCN), as well as in a subset of patients with other leukemias., Methods: This investigation introduced equivalent mutations into the zebrafish csf3r gene via genome editing and used a range of molecular and cellular techniques to understand the impact of these mutations on immune cells across the lifespan., Results: Zebrafish harboring truncated G-CSFRs showed significantly enhanced neutrophil production throughout successive waves of embryonic hematopoiesis and a neutrophil maturation defect in adults, with the mutations acting in a partially dominant manner., Discussion: This study has elucidated new insights into the impact of G-CSFR truncations throughout the life-course and created a bone fide zebrafish model for further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bulleeraz, Goy, Basheer, Liongue and Ward.)

BCL6A is a transcriptional repressor implicated in the development and survival of B and T lymphoctyes, which is also highly expressed in many non-Hodgkin's lymphomas, such as diffuse large B cell lymphoma and follicular lymphoma. Roles in other cell types, including macrophages and non-hematopoietic cells, have also been suggested but require further investigation. This study sought to identify and characterize zebrafish BCL6A and investigate its role in immune cell development and function, with a focus on early macrophages. Bioinformatics analysis identified a homologue for BCL6A ( bcl6aa ), as well as an additional fish-specific duplicate ( bcl6ab ) and a homologue for the closely-related BCL6B ( bcl6b ). The human BCL6A and zebrafish Bcl6aa proteins were highly conserved across the constituent BTB/POZ, PEST and zinc finger domains. Expression of bcl6aa during early zebrafish embryogenesis was observed in the lateral plate mesoderm, a site of early myeloid cell development, with later expression seen in the brain, eye and thymus. Homozygous bcl6aa mutants developed normally until around 14 days post fertilization (dpf), after which their subsequent growth and maturation was severely impacted along with their relative survival, with heterozygous bcl6aa mutants showing an intermediate phenotype. Analysis of immune cell development revealed significantly decreased lymphoid and macrophage cells in both homozygous and heterozygous bcl6aa mutants, being exacerbated in homozygous mutants. In contrast, the number of neutrophils was unaffected. Only the homozygous bcl6aa mutants showed decreased macrophage mobility in response to wounding and reduced ability to contain bacterial infection. Collectively, this suggests strong conservation of BCL6A across evolution, including a role in macrophage biology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Almohaisen, Heidary, Sobah, Ward and Liongue.)

JAK3 is principally activated by members of the interleukin-2 receptor family and plays an essential role in lymphoid development, with inactivating JAK3 mutations causing autosomal-recessive severe combined immunodeficiency (SCID). This study aimed to generate an equivalent zebrafish model of SCID and to characterize the model across the life-course. Genome editing of zebrafish jak3 created mutants similar to those observed in human SCID. Homozygous jak3 mutants showed reduced embryonic T lymphopoiesis that continued through the larval stage and into adulthood, with B cell maturation and adult NK cells also reduced and neutrophils impacted. Mutant fish were susceptible to lymphoid leukemia. This model has many of the hallmarks of human SCID resulting from inactivating JAK3 mutations and will be useful for a variety of pre-clinical applications.

Members of the FOS protein family regulate gene expression responses to a multitude of extracellular signals and are dysregulated in several pathological states. Whilst mouse genetic models have provided key insights into the tissue-specific functions of these proteins in vivo, little is known about their roles during early vertebrate embryonic development. This study examined the potential of using zebrafish as a model for such studies and, more broadly, for investigating the mechanisms regulating the functions of Fos proteins in vivo. Through phylogenetic and sequence analysis, we identified six zebrafish FOS orthologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2 , which show high conservation in key regulatory domains and post-translational modification sites compared to their equivalent human proteins. During embryogenesis, zebrafish fos genes exhibit both overlapping and distinct spatiotemporal patterns of expression in specific cell types and tissues. Most fos genes are also expressed in a variety of adult zebrafish tissues. As in humans, we also found that expression of zebrafish FOS orthologs is induced by oncogenic BRAF-ERK signalling in zebrafish melanomas. These findings suggest that zebrafish represent an alternate model to mice for investigating the regulation and functions of Fos proteins in vertebrate embryonic and adult tissues, and cancer.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Taznin, Perera, Gibert, Ward and Liongue.)

Background: Janus kinase 3 (JAK3) acts downstream of the interleukin-2 (IL-2) receptor family to play a pivotal role in the regulation of lymphoid cell development. Activating JAK3 mutations are associated with a number of lymphoid and other malignancies, with mutations within the regulatory pseudokinase domain common., Methods: The pseudokinase domain mutations A572V and A573V were separately introduced into the highly conserved zebrafish Jak3 and transiently expressed in cell lines and zebrafish embryos to examine their activity and impact on early T cells. Genome editing was subsequently used to introduce the A573V mutation into the zebrafish genome to study the effects of JAK3 activation on lymphoid cells in a physiologically relevant context throughout the life-course., Results: Zebrafish Jak3 A573V produced the strongest activation of downstream STAT5 in vitro and elicited a significant increase in T cells in zebrafish embryos. Zebrafish carrying just a single copy of the Jak3 A573V allele displayed elevated embryonic T cells, which continued into adulthood. Hematopoietic precursors and NK cells were also increased, but not B cells. The lymphoproliferative effects of Jak3 A573V in embryos was shown to be dependent on zebrafish IL-2Rγc, JAK1 and STAT5B equivalents, and could be suppressed with the JAK3 inhibitor Tofacitinib., Conclusions: This study demonstrates that a single JAK3 A573V allele expressed from the endogenous locus was able to enhance lymphopoiesis throughout the life-course, which was mediated via an IL-2Rγc/JAK1/JAK3/STAT5 signaling pathway and was sensitive to Tofacitinib. This extends our understanding of oncogenic JAK3 mutations and creates a novel model to underpin further translational investigations., (© 2022. The Author(s).)