RESUMEN
Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity is shared with human development has not been described. Using single-cell/nucleus RNA sequencing from patient tumors, patient-derived xenografts, primary in vitro cultures, and cell lines, we identify four dominant muscle-lineage cell states: progenitor, proliferative, differentiated, and ground cells. We stratify these RMS cells/nuclei along the continuum of human muscle development and show that they share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that recapitulates fetal muscle development and contain therapy-resistant FN-RMS progenitors that share transcriptomic similarity with bipotent skeletal mesenchymal cells. Fusion-positive RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in myogenic development. This work identifies previously underappreciated cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.
Asunto(s)
Perfilación de la Expresión Génica , Rabdomiosarcoma , Análisis de la Célula Individual , Transcriptoma , Humanos , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Rabdomiosarcoma/metabolismo , Análisis de la Célula Individual/métodos , Animales , Perfilación de la Expresión Génica/métodos , Línea Celular Tumoral , Ratones , Niño , Resistencia a Antineoplásicos/genética , Diferenciación Celular , Desarrollo de Músculos/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , Inmunoterapia , Glicoproteínas de Membrana , Receptores de NetrinaRESUMEN
CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3KAKT- mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.
RESUMEN
Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.
Asunto(s)
Preservación de Órganos , Pez Cebra , Animales , Bioensayo , Técnicas de Cocultivo , Larva , MamíferosRESUMEN
Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.
Asunto(s)
Rabdomiosarcoma Embrionario , Rabdomiosarcoma , Niño , Humanos , Músculo Esquelético/patología , Rabdomiosarcoma/genética , Análisis de la Célula Individual , Células Madre/patologíaRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufficient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly defined. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebrafish and transformation is associated with MYC transcriptional activation. Additionally, we find that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebrafish, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebrafish faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Animales Modificados Genéticamente , Carcinogénesis/metabolismo , Niño , Humanos , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Transducción de Señal/genética , Linfocitos T/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies.
Juvenile myelomonocytic leukaemia is a childhood blood cancer. It is more common in children with a genetic condition called Noonan Syndrome, which causes problems with development in many parts of the body. The most frequent cause is a mutation in a protein called Src homology region 2 domain-containing phosphatase-2, or SHP2 for short. Juvenile myelomonocytic leukaemia starts in the stem cells that normally become blood cells. In children with Noonan Syndrome, these cells show signs of problems before leukaemia begins. Recreating Noonan Syndrome in an animal could shed light on how this childhood cancer develops, but doing this is not straightforward. One option is to use zebrafish, a species of fish in which the embryos are transparent, allowing scientists to watch their blood cells developing under a microscope. They also share many genes with humans, including SHP2. Solman et al. genetically modified zebrafish so they would carry one of the most common mutations seen in children with Noonan Syndrome in the SHP2 protein. The fish had many of the typical features of the condition, including problems producing blood cells. Single cell analysis of the stem cells that become these blood cells showed that, in the mutated fish, these cells had abnormally high levels of activity in genes involved in inflammation. Treating the fish with an anti-inflammatory drug, dexamethasone, reversed the problem. When Solman et al. investigated stem cells from human patients with juvenile myelomonocytic leukaemia, they found the same high levels of activity in inflammatory genes. The current treatment for juvenile myelomonocytic leukaemia is a stem cell transplant, which is only successful in around half of cases. Finding a way to prevent the cancer from developing altogether could save lives. This new line of zebrafish allows researchers to study Noonan Syndrome in more detail, and to test new treatments. A next step could be to find out whether anti-inflammatory drugs have the same effects in mammals as they do in fish.
Asunto(s)
Leucemia Mielomonocítica Juvenil , Síndrome de Noonan , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/metabolismo , Mutación , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Pez CebraRESUMEN
Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Niño , Humanos , Ratones , Animales , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Dasatinib/farmacología , Dasatinib/uso terapéutico , Pez Cebra/metabolismo , Tirosina , Línea Celular Tumoral , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Receptores de Antígenos de Linfocitos T/uso terapéutico , Linfocitos T/metabolismo , Recurrencia , Mamíferos/metabolismoRESUMEN
Antibody-peptide epitope conjugates (APEC) are a new class of modified antibody-drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs. SIGNIFICANCE: This study develops a high-throughput preclinical platform to identify patient-specific antibody-peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.
Asunto(s)
Infecciones por Citomegalovirus , Inmunoconjugados , Neoplasias Ováricas , Animales , Anticuerpos , Linfocitos T CD8-positivos , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Citomegalovirus , Molécula de Adhesión Celular Epitelial , Epítopos , Femenino , Humanos , Inmunoconjugados/uso terapéutico , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Péptido Hidrolasas , Péptidos , Pez CebraRESUMEN
T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single-cell resolution in vivo. Here, optically clear immunocompromised zebrafish were engrafted with fluorescent-labeled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single-cell visualization of T cell-based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement, and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease.
Asunto(s)
Inmunoterapia/métodos , Rabdomiosarcoma/terapia , Análisis de la Célula Individual/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Pez Cebra/genética , Adolescente , Adulto , Animales , Animales Modificados Genéticamente , Niño , Preescolar , Proteínas de Unión al ADN/genética , Receptores ErbB/inmunología , Femenino , Humanos , Inmunoterapia Adoptiva , Subunidad gamma Común de Receptores de Interleucina/genética , Masculino , Ratones Endogámicos , Ftalazinas/farmacología , Piperazinas/farmacología , Rabdomiosarcoma/patología , Linfocitos T/inmunología , Temozolomida/farmacología , Células Tumorales Cultivadas , Proteínas de Pez Cebra/genéticaRESUMEN
Numerous drug treatments that have recently entered the clinic or clinical trials have their genesis in zebrafish. Zebrafish are well established for their contribution to developmental biology and have now emerged as a powerful preclinical model for human disease, as their disease characteristics, aetiology and progression, and molecular mechanisms are clinically relevant and highly conserved. Zebrafish respond to small molecules and drug treatments at physiologically relevant dose ranges and, when combined with cell-specific or tissue-specific reporters and gene editing technologies, drug activity can be studied at single-cell resolution within the complexity of a whole animal, across tissues and over an extended timescale. These features enable high-throughput and high-content phenotypic drug screening, repurposing of available drugs for personalized and compassionate use, and even the development of new drug classes. Often, drugs and drug leads explored in zebrafish have an inter-organ mechanism of action and would otherwise not be identified through targeted screening approaches. Here, we discuss how zebrafish is an important model for drug discovery, the process of how these discoveries emerge and future opportunities for maximizing zebrafish potential in medical discoveries.
Asunto(s)
Ensayos Clínicos como Asunto/estadística & datos numéricos , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Pez CebraRESUMEN
Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in PHF6, encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established in vitro models. Our findings reveal that PHF6 levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of NOTCH1 and its downstream target genes. This functional interaction in developing thymocytes was confirmed in vivo using a phf6-deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced phf6 or notch1 activation. In summary, our work reveals that appropriate control of PHF6 expression is important for normal human hematopoiesis and provides clues towards the role of PHF6 in T-ALL development.
RESUMEN
Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc-/-, il2rgα-/- immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc-/-, il2rgα-/- model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.
Asunto(s)
Transformación Celular Neoplásica , Imagen Molecular/métodos , Análisis de la Célula Individual/métodos , Pez Cebra/inmunología , Animales , Línea Celular Tumoral , HumanosAsunto(s)
Proteínas de Homeodominio/genética , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Animales , Hibridación Genómica Comparativa/métodos , Técnicas de Transferencia de Gen , Genómica/métodos , Pez CebraRESUMEN
In precision oncology, two major strategies are being pursued for predicting clinically relevant tumour behaviours, such as treatment response and emergence of drug resistance: inference based on genomic, transcriptomic, epigenomic and/or proteomic analysis of patient samples, and phenotypic assays in personalized cancer avatars. The latter approach has historically relied on in vivo mouse xenografts and in vitro organoids or 2D cell cultures. Recent progress in rapid combinatorial genetic modelling, the development of a genetically immunocompromised strain for xenotransplantation of human patient samples in adult zebrafish and the first clinical trial using xenotransplantation in zebrafish larvae for phenotypic testing of drug response bring this tiny vertebrate to the forefront of the precision medicine arena. In this Review, we discuss advances in transgenic and transplantation-based zebrafish cancer avatars, and how these models compare with and complement mouse xenografts and human organoids. We also outline the unique opportunities that these different models present for prediction studies and current challenges they face for future clinical deployment.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Pez Cebra , Animales , Modelos Animales de Enfermedad , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Medicina de Precisión , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The mononuclear phagocytic system consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue-resident macrophages, leading to a pressing need for new tools to discriminate mononuclear phagocytic system subsets from other hematopoietic lineages. Macrophage-expressed gene (Mpeg)1.1 is an evolutionary conserved gene encoding perforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since then more than 100 peer-reviewed publications have made use of mpeg1.1-driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation, and function. Whereas the macrophage-specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B-lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1+ lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1+ macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1-positive population in irf8null myeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception is skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the mononuclear phagocytic system in adult zebrafish.