Myelopoiesis during Zebrafish Early Development

Myelopoiesis is the process of producing all types of myeloid cells including monocytes/macrophages and granulocytes.Myeloid cells are known to manifest a wide spectrum of activities such as immune surveillance and tissue remodeling.Irregularities in myeloid cell development and their function are known to associate with the onset and the progression of a variety of human disorders such as leukemia.In the past decades,extensive studies have been carried out in various model organisms to elucidate the molecular mechanisms underlying myelopoiesis with the hope that these efforts will yield knowledge translatable into therapies for related diseases.Zebrafish has recently emerged as a prominent animal model for studying myelopoiesis,especially during early embryogenesis,largely owing to its unique properties such as transparent embryonic body and external development.This review introduces the methodologies used in zebrafish research and focuses on the recent research progresses of zebrafish myelopoiesis.

Zebrafish:a convenient tool for myelopoiesis research

Myelopoiesis is the process in which the mature myeloid cells,including monocytes/macrophages and granulocytes,are developed.Irregular myelopoiesis may cause and deteriorate a variety of hematopoietic malignancies such as leukemia.Myeloid cells and their precursors are difficult to capture in circulation,let alone observe them in real time.For decades,researchers had to face these difficulties,particularly in in-vivo studies.As a unique animal model,zebrafish possesses numerous advantages like body transparency and convenient genetic manipulation,which is very suitable in myelopoiesis research.Here we review current knowledge on the origin and regulation of myeloid development and how zebrafish models were applied in these studies.

Cebpa 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 Cebpa during zebrafish embryonic myelopoiesis. We show that embryonic myelopoiesis in cebpa-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 Cebpa, perhaps acting cooperatively with Runx1, plays a critical role in embryonic neutrophil maintenance. Our findings reveal a new role of Cebpa in embryonic myelopoiesis.

Implications of metabolism-driven myeloid dysfunctions in cancer therapy

Immune homeostasis is maintained by an adequate balance of myeloid and lymphoid responses.In chronic inflammatory states,including cancer,this balance is lost due to dramatic expansion of myeloid progenitors that fail to mature to functional inflammatory neutrophils,macrophages,and dendritic cells(DCs),thus giving rise to a decline in the antitumor effector lymphoid response.Cancer-related inflammation orchestrates the production of hematopoietic growth factors and cytokines that perpetuate recruitment and activation of myeloid precursors,resulting in unresolved and chronic inflammation.This pathologic inflammation creates profound alterations in the intrinsic cellular metabolism of the myeloid progenitor pool,which is amplified by competition for essential nutrients and by hypoxia-induced metabolic rewiring at the tumor site.Therefore,persistent myelopoiesis and metabolic dysfunctions contribute to the development of cancer,as well as to the severity of a broad range of diseases,including metabolic syndrome and autoimmune and infectious diseases.The aims of this review are to(1)define the metabolic networks implicated in aberrant myelopoiesis observed in cancer patients,(2)discuss the mechanisms underlying these clinical manifestations and the impact of metabolic perturbations on clinical outcomes,and(3)explore new biomarkers and therapeutic strategies to restore immunometabolism and differentiation of myeloid cells towards an effector phenotype to increase host antitumor immunity.We propose that the profound metabolic alterations and associated transcriptional changes triggered by chronic and overactivated immune responses in myeloid cells represent critical factors influencing the balance between therapeutic efficacy and immune-related adverse effects(irAEs)for current therapeutic strategies,including immune checkpoint inhibitor(ICI)therapy.

Kzp Regulates the Transcription of gata2 and pu.1 during Primitive Hematopoiesis in Zebrafish Embryos

Kaiso zinc finger-containing protein（Kzp）,a maternally-derived transcription factor,controls dorsoventral patterning during zebrafish gastrulation.Here,we uncovered a new function for Kzp in zebrafish embryonic primitive hematopoiesis.The depletion of kzp led to defects in primitive hematopoiesis including the development of the erythroid and myeloid lineages.On the other hand,overexpression of kzp caused the ectopic expression of gata1,gata2,and pu.1.Chromosome immunoprecipitation assays revealed that Kzp protein directly binds to gata1,gata2,and pu.1 promoters.Interestingly,the ectopic expression of gatal was able to rescue the erythroid,but not the myeloid lineage in kzp-depleted zebrafish embryos.gata1 expression controlled by Kzp was dependent on gatal during primitive erythropoiesis.Our results indicate that Kzp is a critical transcriptional factor for the expression of gata2 and pu.1 to modulate primitive hematopoiesis.

Delineation of a novel dendritic-like subset in human spleen

Dendritic cells （DCs） and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c^hCD11b＋HLA-DR-hCD86＋ subset in the spleen, along with the previously described subsets of hCDlc＋ DCs, hCD123＋ plasmacytoid DCs （pDCs）, hCD16＋ DCs and hCD141＋ DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c＋HLA-DR＋ and hCD11c＋HLA-DR- cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCDSO and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors.