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.

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.

Two phenotypically and functionally distinct microglial populations in adult zebrafish.

Microglia are the tissue-resident macrophages in the central nervous system and are critically involved in immune defense, neural development and function, and neuroinflammation. The versatility of microglia has long been attributed to heterogeneity. Recent studies have revealed possible heterogeneity in human but not in murine microglia, yet a firm demonstration linking microglial heterogeneity to functional phenotypes remains scarce. Here, we identified two distinct microglial populations in adult zebrafish that differ in morphology, distribution, development, and function. The predominant population, phagocytotic microglia, which expresses ccl34b.1, is broadly distributed, amoeboid in shape, highly mobile, and phagocytotic. The other white matter-enriched ccl34b.1- population, regulatory microglia, has ramified protrusions but has limited mobility and phagocytosis capability. These functional differences are further supported by distinct transcriptomes and responses to bacterial infection, where ccl34b.1+ microglia function in tissue clearance and ccl34b.1- microglia release immune regulators. Our study sheds light on the heterogeneity and functional diversification of microglia.

Dysregulation of Microglial Function Contributes to Neuronal Impairment in Mcoln1a-Deficient Zebrafish.

Type IV mucolipidosis (ML-IV) is a neurodegenerative lysosome storage disorder caused by mutations in the MCOLN1 gene. However, the cellular and molecular bases underlying the neuronal phenotypes of ML-IV disease remain elusive. Using a forward genetic screening, we identified a zebrafish mutant, biluo, that harbors a hypomorphic mutation in mcoln1a, one of the two zebrafish homologs of mammalian MCOLN1. The mcoln1a-deficient mutants display phenotypes partially recapitulating the key features of ML-IV disorder, including the accumulation of enlarged late endosomes in microglia and aberrant neuronal activities in both spontaneous and visual-evoking conditions in optic tectal neurons. We further show that the accumulation of enlarged late endosomes in microglia is caused by the impairment of late endosome and lysosome fusion and the aberrant neuronal activities can be partially rescued by the reconstitution of Mcoln1a function in microglia. Our findings suggest that dysregulation of microglial function may contribute to the development and progression of ML-IV disease.

The first wave of T lymphopoiesis in zebrafish arises from aorta endothelium independent of hematopoietic stem cells.

T lymphocytes are key cellular components of the adaptive immune system and play a central role in cell-mediated immunity in vertebrates. Despite their heterogeneities, it is believed that all different types of T lymphocytes are generated exclusively via the differentiation of hematopoietic stem cells (HSCs). Using temporal-spatial resolved fate-mapping analysis and time-lapse imaging, here we show that the ventral endothelium in the zebrafish aorta-gonad-mesonephros and posterior blood island, the hematopoietic tissues previously known to generate HSCs and erythromyeloid progenitors, respectively, gives rise to a transient wave of T lymphopoiesis independent of HSCs. This HSC-independent T lymphopoiesis occurs early and generates predominantly CD4 Tαß cells in the larval but not juvenile and adult stages, whereas HSC-dependent T lymphopoiesis emerges late and produces various subtypes of T lymphocytes continuously from the larval stage to adulthood. Our study unveils the existence, origin, and ontogeny of HSC-independent T lymphopoiesis in vivo and reveals the complexity of the endothelial-hematopoietic transition of the aorta.

Cebpα is essential for the embryonic myeloid progenitor and neutrophil maintenance in zebrafish.

In vertebrates, myeloid cells arise from multiple waves of development: the first or embryonic wave of myelopoiesis initiates early from non-hematopoietic stem cell (HSC) precursors and gives rise to myeloid cells transiently during early development; whereas the second or adult wave of myelopoiesis emerges later from HSCs and produces myeloid cells continually during fetal and adult life. In the past decades, a great deal has been learnt about the development of myeloid cells from adult myelopoiesis, yet the genetic network governing embryonic myelopoiesis remains poorly defined. In this report, we present an in vivo study to delineate the role of Cebpα during zebrafish embryonic myelopoiesis. We show that embryonic myelopoiesis in cebpα-deficient zebrafish mutants initiates properly but fails to produce macrophages and neutrophils. The lack of macrophages and neutrophils in the mutants is largely attributed to the cell cycle arrest of embryonic myeloid progenitors, resulting in the impairment of their maintenance and subsequent differentiation. We further show that Cebpα, perhaps acting cooperatively with Runx1, plays a critical role in embryonic neutrophil maintenance. Our findings reveal a new role of Cebpα in embryonic myelopoiesis.