Macrophages in tissue repair and regeneration: insights from zebrafish.

Macrophages play crucial and versatile roles in regulating tissue repair and regeneration upon injury. However, due to their complex compositional heterogeneity and functional plasticity, deciphering the nature of different macrophage subpopulations and unraveling their dynamics and precise roles during the repair process have been challenging. With its distinct advantages, zebrafish (Danio rerio) has emerged as an invaluable model for studying macrophage development and functions, especially in tissue repair and regeneration, providing valuable insights into our understanding of macrophage biology in health and diseases. In this review, we present the current knowledge and challenges associated with the role of macrophages in tissue repair and regeneration, highlighting the significant contributions made by zebrafish studies. We discuss the unique advantages of the zebrafish model, including its genetic tools, imaging techniques, and regenerative capacities, which have greatly facilitated the investigation of macrophages in these processes. Additionally, we outline the potential of zebrafish research in addressing the remaining challenges and advancing our understanding of the intricate interplay between macrophages and tissue repair and regeneration.

Styxl2 regulates de novo sarcomere assembly by binding to non-muscle myosin IIs and promoting their degradation.

Styxl2, a poorly characterized pseudophosphatase, was identified as a transcriptional target of the Jak1-Stat1 pathway during myoblast differentiation in culture. Styxl2 is specifically expressed in vertebrate striated muscles. By gene knockdown in zebrafish or genetic knockout in mice, we found that Styxl2 plays an essential role in maintaining sarcomere integrity in developing muscles. To further reveal the functions of Styxl2 in adult muscles, we generated two inducible knockout mouse models: one with Styxl2 being deleted in mature myofibers to assess its role in sarcomere maintenance, and the other in adult muscle satellite cells (MuSCs) to assess its role in de novo sarcomere assembly. We find that Styxl2 is not required for sarcomere maintenance but functions in de novo sarcomere assembly during injury-induced muscle regeneration. Mechanistically, Styxl2 interacts with non-muscle myosin IIs, enhances their ubiquitination, and targets them for autophagy-dependent degradation. Without Styxl2, the degradation of non-muscle myosin IIs is delayed, which leads to defective sarcomere assembly and force generation. Thus, Styxl2 promotes de novo sarcomere assembly by interacting with non-muscle myosin IIs and facilitating their autophagic degradation.

The different roles of V-ATPase a subunits in phagocytosis/endocytosis and autophagy.

Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species.Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.

Interplay of Zeb2a, Id2a and Batf3 regulates microglia and dendritic cell development in the zebrafish brain.

In vertebrates, the central nervous system (CNS) harbours various immune cells, including parenchymal microglia, perivascular macrophages and dendritic cells, which act in coordination to establish an immune network to regulate neurogenesis and neural function, and to maintain the homeostasis of the CNS. Recent single cell transcriptomic profiling has revealed that the adult zebrafish CNS contains microglia, plasmacytoid dendritic cells (pDCs) and two conventional dendritic cells (cDCs), ccl35+ cDCs and cnn3a+cDCs. However, how these distinct myeloid cells are established in the adult zebrafish CNS remains incompletely defined. Here, we show that the Inhibitor of DNA binding 2a (Id2a) is essential for the development of pDCs and cDCs but is dispensable for the formation of microglia, whereas the Basic leucine zipper transcription factor ATF-like 3 (Batf3) acts downstream of id2a and is required exclusively for the formation of the cnn3a+ cDC subset. In contrast, the Zinc finger E-box-binding homeobox 2a (Zeb2a) promotes the expansion of microglia and inhibits the DC specification, possibly through repressing id2a expression. Our study unravels the genetic networks that govern the development of microglia and brain-associated DCs in the zebrafish CNS.

Sulindac selectively induces autophagic apoptosis of GABAergic neurons and alters motor behaviour in zebrafish.

Nonsteroidal anti-inflammatory drugs compose one of the most widely used classes of medications, but the risks for early development remain controversial, especially in the nervous system. Here, we utilized zebrafish larvae to assess the potentially toxic effects of nonsteroidal anti-inflammatory drugs and found that sulindac can selectively induce apoptosis of GABAergic neurons in the brains of zebrafish larvae brains. Zebrafish larvae exhibit hyperactive behaviour after sulindac exposure. We also found that akt1 is selectively expressed in GABAergic neurons and that SC97 (an Akt1 activator) and exogenous akt1 mRNA can reverse the apoptosis caused by sulindac. Further studies showed that sulindac binds to retinoid X receptor alpha (RXRα) and induces autophagy in GABAergic neurons, leading to activation of the mitochondrial apoptotic pathway. Finally, we verified that sulindac can lead to hyperactivity and selectively induce GABAergic neuron apoptosis in mice. These findings suggest that excessive use of sulindac may lead to early neurodevelopmental toxicity and increase the risk of hyperactivity, which could be associated with damage to GABAergic neurons.

Safety of high-carbohydrate fluid diet 2 h versus overnight fasting before non-emergency endoscopic retrograde cholangiopancreatography: A single-blind, multicenter, randomized controlled trial.

BACKGROUND: Although overnight fasting is recommended prior to endoscopic retrograde cholangiopancreatography (ERCP), the benefits and safety of high-carbohydrate fluid diet (CFD) intake 2 h before ERCP remain unclear. This study aimed to analyze whether high-CFD intake 2 h before ERCP can be safe and accelerate patients' recovery. METHODS: This prospective, multicenter, randomized controlled trial involved 15 tertiary ERCP centers. A total of 1330 patients were randomized into CFD group (n = 665) and fasting group (n = 665). The CFD group received 400 mL of maltodextrin orally 2 h before ERCP, while the control group abstained from food/water overnight (>6 h) before ERCP. All ERCP procedures were performed using deep sedation with intravenous propofol. The investigators were blinded but not the patients. The primary outcomes included postoperative fatigue and abdominal pain score, and the secondary outcomes included complications and changes in metabolic indicators. The outcomes were analyzed according to a modified intention-to-treat principle. RESULTS: The post-ERCP fatigue scores were significantly lower at 4 h (4.1 ± 2.6 vs. 4.8 ± 2.8, t = 4.23, P <0.001) and 20 h (2.4 ± 2.1 vs. 3.4 ± 2.4, t = 7.94, P <0.001) in the CFD group, with least-squares mean differences of 0.48 (95% confidence interval [CI]: 0.26-0.71, P <0.001) and 0.76 (95% CI: 0.57-0.95, P <0.001), respectively. The 4-h pain scores (2.1 ± 1.7 vs. 2.2 ± 1.7, t = 2.60, P = 0.009, with a least-squares mean difference of 0.21 [95% CI: 0.05-0.37]) and positive urine ketone levels (7.7% [39/509] vs. 15.4% [82/533], χ2 = 15.13, P <0.001) were lower in the CFD group. The CFD group had significantly less cholangitis (2.1% [13/634] vs. 4.0% [26/658], χ2 = 3.99, P = 0.046) but not pancreatitis (5.5% [35/634] vs. 6.5% [43/658], χ2 = 0.59, P = 0.444). Subgroup analysis revealed that CFD reduced the incidence of complications in patients with native papilla (odds ratio [OR]: 0.61, 95% CI: 0.39-0.95, P = 0.028) in the multivariable models. CONCLUSION: Ingesting 400 mL of CFD 2 h before ERCP is safe, with a reduction in post-ERCP fatigue, abdominal pain, and cholangitis during recovery. TRAIL REGISTRATION: ClinicalTrials.gov, No. NCT03075280.

Cross-organ single-cell transcriptome profiling reveals macrophage and dendritic cell heterogeneity in zebrafish.

Tissue-resident macrophages (TRMs) and dendritic cells (DCs) are highly heterogeneous and essential for immunity, tissue regeneration, and homeostasis maintenance. Here, we comprehensively profile the heterogeneity of TRMs and DCs across adult zebrafish organs via single-cell RNA sequencing. We identify two macrophage subsets: pro-inflammatory macrophages with potent phagocytosis signatures and pro-remodeling macrophages with tissue regeneration signatures in barrier tissues, liver, and heart. In parallel, one conventional dendritic cell (cDC) population with prominent antigen presentation capacity and plasmacytoid dendritic cells (pDCs) featured by anti-virus properties are also observed in these organs. Remarkably, in addition to a single macrophage/microglia population with potent phagocytosis capacity, a pDC population and two distinct cDC populations are identified in the brain. Finally, we generate specific reporter lines for in vivo tracking of macrophage and DC subsets. Our study depicts the landscape of TRMs and DCs and creates valuable tools for in-depth study of these cells in zebrafish.

Cell competition for neuron-derived trophic factor controls the turnover and lifespan of microglia.

Microglia are brain-resident macrophages capable of long-term maintenance through self-renewal. Yet the mechanism governing the turnover and lifespan of microglia remains unknown. In zebrafish, microglia arise from two sources, rostral blood island (RBI) and aorta-gonad-mesonephros (AGM). The RBI-derived microglia are born early but have a short lifespan and diminish in adulthood, while the AGM-derived microglia emerge later and are capable of long-term maintenance in adulthood. Here, we show that the attenuation of RBI microglia is due to their less competitiveness for neuron-derived interleukin-34 (Il34) caused by age-dependent decline of colony-stimulating factor-1 receptor a (csf1ra). Alterations of Il34/Csf1ra levels and removal of AGM microglia revamp the proportion and lifespan of RBI microglia. The csf1ra/CSF1R expression in zebrafish AGM-derived microglia and murine adult microglia also undergo age-dependent decline, leading to the elimination of aged microglia. Our study reveals cell competition as a general mechanism controlling the turnover and lifespan of microglia.

Metaphocytes are IL-22BP-producing cells regulated by ETS transcription factor Spic and essential for zebrafish barrier immunity.

Metaphocytes are tissue-resident macrophage (TRM)/dendritic cell (DC)-like cells of non-hematopoietic origin in zebrafish barrier tissues. One remarkable property of metaphocytes is their ability to capture soluble antigens from the external environment via transepithelial protrusions, a unique function manifested by specialized subpopulations of the TRMs/DCs in mammal barrier tissues. Yet, how metaphocytes acquire myeloid-like cell properties from non-hematopoietic precursors and how they regulate barrier immunity remains unknown. Here, we show that metaphocytes are in situ generated from local progenitors guided by the ETS transcription factor Spic, the deficiency of which results in the absence of metaphocytes. We further document that metaphocytes are the major IL-22BP-producing cells, and the depletion of metaphocytes causes dysregulated barrier immunity that resembles the phenotype of IL-22BP-deficient mice. These findings reveal the ontogeny, development, and function of metaphocytes in zebrafish, which facilitates our understanding of the nature and function of the mammalian TRM/DC counterparts.

Learning from Zebrafish Hematopoiesis.

Hematopoiesis is a complex process that tightly regulates the generation, proliferation, differentiation, and maintenance of hematopoietic cells. Disruptions in hematopoiesis can lead to various diseases affecting both hematopoietic and non-hematopoietic systems, such as leukemia, anemia, thrombocytopenia, rheumatoid arthritis, and chronic granuloma. The zebrafish serves as a powerful vertebrate model for studying hematopoiesis, offering valuable insights into both hematopoietic regulation and hematopoietic diseases. In this chapter, we present a comprehensive overview of zebrafish hematopoiesis, highlighting its distinctive characteristics in hematopoietic processes. We discuss the ontogeny and modulation of both primitive and definitive hematopoiesis, as well as the microenvironment that supports hematopoietic stem/progenitor cells. Additionally, we explore the utility of zebrafish as a disease model and its potential in drug discovery, which not only advances our understanding of the regulatory mechanisms underlying hematopoiesis but also facilitates the exploration of novel therapeutic strategies for hematopoietic diseases.

The ETS transcription factor Spi2 regulates hematopoietic cell development in zebrafish.

The E26 transformation-specific or E-twenty-six (ETS) genes encode a superfamily of transcription factors involved in diverse biological processes. Here, we report the identification and characterization of a previously unidentified member of the ETS transcription factors, Spi2, that is found exclusively in the ray-finned fish kingdom. We show that the expression of spi2 is restricted to hemogenic endothelial cells (HECs) and to hematopoietic stem and progenitor cells (HSPCs) in zebrafish. Using bacteria artificial chromosome transgenesis, we generate a spi2 reporter line, TgBAC(spi2:P2a-GFP), which manifests the GFP pattern recapitulating the endogenous spi2 expression. Genetic ablation of spi2 has little effect on HEC formation and the endothelial-to-hematopoietic transition, but results in compromised proliferation of HSPCs in the caudal hematopoietic tissue (CHT) during early development and in severe myeloid lineage defect in adulthood. Epistatic analysis shows that spi2 acts downstream of runx1 in regulating HSPC development in the CHT. Our study identifies Spi2 as an essential regulator for definitive hematopoietic cell development and creates a TgBAC(spi2:P2a-GFP) reporter line for tracking HECs, HSPCs, myeloid cells and thrombocytes from early development to adulthood.

Mafba and Mafbb regulate microglial colonization of zebrafish brain via controlling chemotaxis receptor expression.

Microglia are the central nervous system (CNS)-resident macrophages involved in neural inflammation, neurogenesis, and neural activity regulation. Previous studies have shown that naturally occurring neuronal apoptosis plays a critical role in regulating microglial colonization of the brain in zebrafish. However, the molecular signaling cascades underlying neuronal apoptosis-mediated microglial colonization and the regulation of these cascades remain undefined. Here, we show that basic leucine zipper (b-Zip) transcription factors, Mafba and Mafbb, two zebrafish orthologs of mammalian MAFB, are key regulators in neuronal apoptosis-mediated microglial colonization of the brain in zebrafish. We document that the loss of Mafba and Mafbb function perturbs microglial colonization of the brain. We further demonstrate that Mafba and Mafbb act cell-autonomously and cooperatively to orchestrate microglial colonization, at least in part, by regulating the expression of G protein-coupled receptor 34a (Gpr34a), which directs peripheral macrophage recruitment into the brain through sensing the lysophosphatidylserine (lysoPS) released by the apoptotic neurons. Our study reveals that Mafba and Mafbb regulate neuronal apoptosis-mediated microglial colonization of the brain in zebrafish via the lysoPS-Gpr34a pathway.

Csf1rb regulates definitive hematopoiesis in zebrafish.

In vertebrates, hematopoietic stem and progenitor cells (HSPCs) are capable of self-renewal and continuously replenishing all mature blood lineages throughout life. However, the molecular signaling regulating the maintenance and expansion of HSPCs remains incompletely understood. Colony-stimulating factor 1 receptor (CSF1R) is believed to be the primary regulator for the myeloid lineage but not HSPC development. Here, we show a surprising role of Csf1rb, a zebrafish homolog of mammalian CSF1R, in preserving the HSPC pool by maintaining the proliferation of HSPCs. Deficiency of csf1rb leads to a reduction in both HSPCs and their differentiated progenies, including myeloid, lymphoid and erythroid cells at early developmental stages. Likewise, the absence of csf1rb conferred similar defects upon HSPCs and leukocytes in adulthood. Furthermore, adult hematopoietic cells from csf1rb mutants failed to repopulate immunodeficient zebrafish. Interestingly, loss-of-function and gain-of-function assays suggested that the canonical ligands for Csf1r in zebrafish, including Csf1a, Csf1b and Il34, were unlikely to be ligands of Csf1rb. Thus, our data indicate a previously unappreciated role of Csf1r in maintaining HSPCs, independently of known ligands.

Hemogenic and aortic endothelium arise from a common hemogenic angioblast precursor and are specified by the Etv2 dosage.

SignificanceHematopoietic stem cells (HSCs) are generated from specialized endothelial cells, called hemogenic endothelial cells (HECs). It has been debated whether HECs and non-HSC-forming conventional endothelial cells (cECs) arise from a common precursor or represent distinct lineages. Moreover, the molecular basis underlying their distinct fate determination is poorly understood. We use photoconvertible labeling, time-lapse imaging, and single-cell RNA-sequencing analysis to trace the lineage of HECs. We discovered that HECs and cECs arise from a common hemogenic angioblast precursor, and their distinct fate is determined by high or low dosage of Etv2, respectively. Our results illuminate the lineage origin and a mechanism on the fate determination of HECs, which may enhance the understanding on the ontogeny of HECs in vertebrates.

Tumor-associated macrophages promote cholangiocarcinoma progression via exosomal Circ_0020256.

This study investigated the exosomal circular RNAs (CircRNAs) produced by tumor-associated macrophages and delivered into the microenvironment of cholangiocarcinoma cells in order to use them as molecular targets for clinical therapy. Tumor-associated M2 macrophages (TAMs) were induced from THP-1 cells and identified by flow cytometry. The TAM-secreted exosomes were isolated from conditioned medium and a CircRNA microarray assay was performed to identify CircRNAs that were uniquely expressed in the isolated exosomes. Circ_0020256 was especially identified based on having the highest differential expression level among all of the CircRNA candidates. In vitro and in vivo experiments were performed to assess the effects of TAMs, exosomes, and Circ_0020256 on the growth and migration of cholangiocarcinoma (CCA) cells. The induced TAMs promoted the proliferation, migration, and invasion of CCA cells and those effects were mediated by exosomes secreted by the TAMs. In CCA cells (RBE and HCCC-9810), Circ_0020256 significantly promoted cellular activity by interacting with its intra-cellular microRNA target, miR-432-5p. In contrast, overexpression of transcription factor E2F3 in CCA cells restored the CCA cellular activities that were inhibited by miR-432-5p. On the other hand, treatment with small interference RNA (siRNA) for Circ_0020256 inhibited CCA cell proliferation, migration, and invasion both in vitro and in vivo. In conclusion, Circ_0020256 in TAM-secreted exosomes promoted the proliferation, migration, and invasion of CCA cells, and that promotional activity was regulated via a Circ_0020256/miR-432-5p/E2F3 axis.

Identification of resection plane for anatomical liver resection using ultrasonography-guided needle insertion.

Purposes: To set up an easy-handled and precise delineation of resection plane for hepatic anatomical resection (AR). Methods: Cases of AR using ultrasonography-guided needle insertion to trace the target hepatic vein for delineation of resection planes [new technique (NT) group, n = 22] were retrospectively compared with those without implementation of this surgical technique [traditional technique (TT) group, n = 29] in terms of perioperative courses and surgical outcomes. Results: The target hepatic vein was successfully exposed in all patients of the NT group, compared with a success rate of 79.3% in the TT group (P < 0.05). The average operation time and intraoperative blood loss were 280 ± 32 min and 550 ± 65 ml, respectively, in the NT group. No blood transfusion was required in either group. The postoperative morbidities (bile leakage and peritoneal effusion) were similar between groups. No mortality within 90 days was observed. Conclusions: Ultrasonography-guided needle insertion is a convenient, safe and efficient surgical approach to define a resection plane for conducting AR.

The spliceosome factor sart3 regulates hematopoietic stem/progenitor cell development in zebrafish through the p53 pathway.

Hematopoietic stem cells (HSCs) possess the potential for self-renew and the capacity, throughout life, to differentiate into all blood cell lineages. Yet, the mechanistic basis for HSC development remains largely unknown. In this study, we characterized a zebrafish smu471 mutant with hematopoietic stem/progenitor cell (HSPC) defects and found that sart3 was the causative gene. RNA expression profiling of the sart3smu471 mutant revealed spliceosome and p53 signaling pathway to be the most significantly enriched pathways in the sart3smu471 mutant. Knock down of p53 rescued HSPC development in the sart3smu471 mutant. Interestingly, the p53 inhibitor, mdm4, had undergone an alternative splicing event in the mutant. Restoration of mdm4 partially rescued HSPC deficiency. Thus, our data suggest that HSPC proliferation and maintenance require sart3 to ensure the correct splicing and expression of mdm4, so that the p53 pathway is properly inhibited to prevent definitive hematopoiesis failure. This study expands our knowledge of the regulatory mechanisms that impact HSPC development and sheds light on the mechanistic basis and potential therapeutic use of sart3 in spliceosome-mdm4-p53 related disorders.

De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

[Figure: see text].

Slc20a1b is essential for hematopoietic stem/progenitor cell expansion in zebrafish.

Hematopoietic stem and progenitor cells (HSPCs) are able to self-renew and can give rise to all blood lineages throughout their lifetime, yet the mechanisms regulating HSPC development have yet to be discovered. In this study, we characterized a hematopoiesis defective zebrafish mutant line named smu07, which was obtained from our previous forward genetic screening, and found the HSPC expansion deficiency in the mutant. Positional cloning identified that slc20a1b, which encodes a sodium phosphate cotransporter, contributed to the smu07 blood phenotype. Further analysis demonstrated that mutation of slc20a1b affects HSPC expansion through cell cycle arrest at G2/M phases in a cell-autonomous manner. Our study shows that slc20a1b is a vital regulator for HSPC proliferation in zebrafish early hematopoiesis and provides valuable insights into HSPC development.