The Ontogeny of Definitive Hematopoiesis in the Zebrafish.

Abstract Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. The generation of definitive, or multilineage, hematopoietic precursors during embryogenesis remains less well understood. Here we show, using a combination of gene expression analyses, prospective isolation approaches, hematopoietic progenitor cultures, transplantation, and in vivo lineage tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of hematopoietic stem cells (HSCs). EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. By contrast, fate mapping studies of CD41:eGFP+ cells residing in the aorta/gonads/mesonephros (AGM) region demonstrate robust colonization of the pronephros and thymus. Using timelapse microscopy, we show that these sites of adult hematopoiesis are seeded by CD41+ cells that migrate along the pronephric ducts from the AGM. These studies provide phenotypic and functional analyses of the first hematopoietic stem and progenitor cells in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.

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The Ontogeny of Definitive Hematopoiesis in the Zebrafish.

Blood ◽

10.1182/blood.v112.11.sci-43.sci-43 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-43-sci-43

Author(s):

Julien Bertrand ◽

Albert Kim ◽

Jennifer Cisson ◽

David Stachura ◽

David Traver

Keyword(s):

Progenitor Cells ◽

Functional Characterization ◽

Time Lapse ◽

Notch Receptor ◽

Proliferative Potential ◽

Fate Mapping ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Definitive Hematopoiesis

Abstract Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed “primitive” and “definitive.” Primitive hematopoiesis rapidly generates erythrocytes and macrophages through monopotent precursors for immediate use in the developing embryo. Definitive hematopoiesis arises later and generates multipotent precursors, including hematopoietic stem cells (HSCs). We have recently performed the first prospective isolation and functional characterization of hematopoietic stem and progenitor cells in the zebrafish and shown that definitive hematopoiesis generates two distinct precursor types during embryogenesis. First to arise are erythromyeloid progenitors (EMPs) in the posterior blood island (PBI), cells that possess robust but transient proliferation potential but lack self-renewal and lymphoid differentiation capacities. Next to develop are HSCs in the aorta/gonad/mesonephros (AGM) region. Unlike EMPs, HSCs colonize the developing thymus to initiate T-lymphopoiesis and seed the pronephros, the site of adult hematopoiesis. In vivo fate mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. By contrast, fate mapping of CD41:eGFP+ cells residing in the AGM region demonstrate robust colonization of the pronephros and thymus. Using time-lapse microscopy, we have observed directly the behaviors of the first HSCs to arise in the embryo. AGM HSCs, marked by a CD41:eGFP or c-myb:eGFP transgene, enter circulation to seed the thymic anlage and migrate along the pronephric tubules to seed the pronephros. We are currently performing retrospective time-lapse analyses to determine where in the early embryo HSC and EMP precursors are born. These data will be informative in analyzing where instructive ligands are expressed, including the Delta genes that provide paracrine signals to cells expressing the Notch receptor. We have demonstrated that Notch signaling is necessary for the generation of HSCs but dispensable for EMP formation. We have utilized new and existing Notch reporter lines to determine more precisely where and when HSC precursors receive Notch signals. Together, these studies highlight the power of the zebrafish system in combining genetic approaches with the direct imaging of hematopoietic stem and progenitor cells in living embryos.

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Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

10.21203/rs.3.rs-1034533/v1 ◽

2021 ◽

Author(s):

Xu Fan ◽

Pei Lu ◽

Xianghua Cui ◽

Peng Wu ◽

Weiran Lin ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Kupffer Cells ◽

Lineage Tracing ◽

Genetic Lineage ◽

Fate Mapping ◽

Monocytic Cell ◽

Hematopoietic Stem ◽

Cellular Origin

Abstract Kupffer cells (KCs) originate from yolk sac progenitors before birth, but the origin of repopulating KCs in adult remains unclear. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells, which present a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

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Kinetics of adult hematopoietic stem cell differentiation in vivo

Journal of Experimental Medicine ◽

10.1084/jem.20180136 ◽

2018 ◽

Vol 215 (11) ◽

pp. 2815-2832 ◽

Cited By ~ 23

Author(s):

Samik Upadhaya ◽

Catherine M. Sawai ◽

Efthymia Papalexi ◽

Ali Rashidfarrokhi ◽

Geunhyo Jang ◽

...

Keyword(s):

Stem Cell Differentiation ◽

Cell Types ◽

Lineage Tracing ◽

Intermediate Cell ◽

Hematopoietic Stem ◽

Hematopoietic Stem Cell Differentiation ◽

Kinetics Of ◽

Kinetics In Vivo

Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.

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Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development

Blood ◽

10.1182/blood-2007-01-069005 ◽

2007 ◽

Vol 109 (12) ◽

pp. 5208-5214 ◽

Cited By ~ 94

Author(s):

Hao Jin ◽

Jin Xu ◽

Zilong Wen

Keyword(s):

Progenitor Cells ◽

Fetal Liver ◽

Embryo Cell ◽

Migration Route ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Blood Island ◽

Adult Kidney ◽

Definitive Hematopoiesis

Abstract The development of vertebrate definitive hematopoiesis is featured by temporally and spatially dynamic distribution of hematopoietic stem/progenitor cells (HSPCs). It is proposed that the migration of definitive HSPCs, at least in part, accounts for this unique characteristic; however, compelling in vivo lineage evidence is still lacking. Here we present an in vivo analysis to delineate the migration route of definitive HSPCs in the early zebrafish embryo. Cell-marking analysis was able to first map definitive HSPCs to the ventral wall of dorsal aorta (DA). These cells were subsequently found to migrate to a previously unappreciated organ, posterior blood island (PBI), located between the caudal artery and caudal vein, and finally populate the kidney, the adult hematopoietic organ. These findings demonstrate that the PBI acts as an intermediate hematopoietic organ in a manner analogous to the mammalian fetal liver to sustain definitive hematopoiesis before adult kidney hematopoiesis occurs. Thus our study unambiguously documents the in vivo trafficking of definitive HSPCs among developmentally successive hematopoietic compartments and underscores the ontogenic conservation of definitive hematopoiesis between zebrafish and mammals.

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Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

10.1101/2020.07.31.230649 ◽

2020 ◽

Author(s):

Xu Fan ◽

Pei Lu ◽

Xianghua Cui ◽

Peng Wu ◽

Weiran Lin ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Kupffer Cells ◽

Lineage Tracing ◽

Genetic Lineage ◽

Fate Mapping ◽

Monocytic Cell ◽

Hematopoietic Stem ◽

Cellular Origin

AbstractKupffer cells (KCs) originate from yolk sac progenitors before birth. Throughout adulthood, they self-maintain independently from the input of circulating monocytes (MOs) at stead state, and are replenished within 2 weeks after having been depleted, but the origin of repopulating KCs in adult remains unclear. The current paradigm dictates that repopulating KCs originate from preexisting KCs or monocytes, but there remains a lack of fate-mapping evidence. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided strong in vivo fate-mapping evidences that repopulating KCs originate directly from Hematopoietic stem cells not from preexisting KCs or from MOs.SignificanceThere is a standing controversy in the field regarding the cellular origin of repopulating macrophages. This paper provides strong in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells not from preexisting KCs or from MOs, which presenting a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

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Zebrafish Growth Factor Independence Transcription Factors Establish a New Paradigm for Regulation of Primitive and Definitive Hematopoietic Lineages,

Blood ◽

10.1182/blood.v118.21.3379.3379 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3379-3379

Author(s):

Jeffrey D. Cooney ◽

Ebrahim Shafizadeh ◽

Paul F. McBride ◽

Kelli J. Carroll ◽

Heidi Anderson ◽

...

Keyword(s):

Transcription Factors ◽

Growth Factor ◽

Essential Role ◽

Cell Mass ◽

Intermediate Cell ◽

New Paradigm ◽

Hematopoietic Stem ◽

Isolation And Characterization ◽

Definitive Hematopoiesis

Abstract Abstract 3379 The Growth Factor Independence (Gfi) zinc finger transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic lineage specification. In mammals, Gfi1 regulates hematopoietic stem cell (HSC) and lymphoid populations, while Gfi1b is required for megakaryocyte and erythroid development (van der Meer, et al. 2010 Leukemia 11:1834–43). In zebrafish, gfi1.1 plays an essential role in primitive hematopoiesis, preserving primitive HSC populations and regulating the erythroid-myeloid balance (Wei, et al. 2008 Cell Res. 6:677–85). However, little is known about the role of gfi1.1 in definitive hematopoiesis or about the role of additional hematopoietic gfi family members in zebrafish. Here, we report the isolation and characterization of an additional zebrafish gfi family transcription factor, gfi1.2b. We compare and contrast gfi1.1 and gfi1.2b, showing that they are highly expressed in the intermediate cell mass (ICM) and aorta-gonad-mesonephros (AGM), the respective sites of primitive and definitive hematopoiesis in zebrafish. Using antisense morpholino oligos (MO), whole mount in situ hybridization (WISH) and fluorescent activated cell sorting (FACS) of transgenic reporter fish, we demonstrate that gfi1.1 and gfi1.2b have distinct, essential roles in preserving primitive and definitive HSC populations, respectively. Loss of gfi1.1 specifically silences expression of scl and gata-1, markers of primitive HSC and erythroid progenitors. Conversely, loss of gfi1.2b silences expression of Tg(cd41:eGFPlo) cells, indicating an essential role for gfi1.2b in preserving definitive hematopoietic progenitors (Ma, et al. 2011 Blood 118:289–297). Consistent with the discrete roles of gfi1.1 and gfi1.2b in primitive and definitive lineages, knockdown of gfi1.2b silences lymphocyte rag-1 expression in the developing thymus, while knockdown of gfi1.1 has no effect on the thymic lymphocyte population. gfi1.1 and gfi1.2b have overlapping roles in erythropoiesis, as loss of either gfi factor reduces erythrocyte populations, while loss of both gfi paralogs results in a more profound silencing of erythrocytes. We further demonstrate that loss of gata-1 reduces gfi1.1 expression and silences gfi1.2b, suggesting that gata-1 plays an essential role in regulating the transcription of both genes. Together, these studies demonstrate that gfi1.1 and gfi1.2b have distinct and overlapping roles in zebrafish hematopoiesis and establish a new paradigm for the regulation of primitive and definitive hematopoietic lineages by gfi transcription factors. Disclosures: No relevant conflicts of interest to declare.

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A Xenopus homologue of aml-1 reveals unexpected patterning mechanisms leading to the formation of embryonic blood

Development ◽

10.1242/dev.125.8.1371 ◽

1998 ◽

Vol 125 (8) ◽

pp. 1371-1380 ◽

Cited By ~ 5

Author(s):

W.D. Tracey ◽

M.E. Pepling ◽

M.E. Horb ◽

G.H. Thomsen ◽

J.P. Gergen

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Lineage Tracing ◽

Runt Domain ◽

Hematopoietic Stem ◽

Blood Island ◽

Primitive Hematopoiesis ◽

Vertebrate Embryos ◽

Definitive Hematopoiesis

The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. We have isolated Xaml, a Xenopus AML1 homologue in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventral blood island in a pattern that anticipates that of later globin. Analysis of globin and Xaml expression in explants, in embryos with perturbed dorsal ventral patterning, and by lineage tracing indicates that the formation of the ventral blood island is more complex than previously thought and involves contributions from both dorsal and ventral tissues. A truncated Xaml protein interferes with primitive hematopoiesis. Based on these results, we propose that Runt domain proteins function in the specification of hematopoietic stem cells in vertebrate embryos.

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Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽

10.1182/blood.v74.3.930.930 ◽

1989 ◽

Vol 74 (3) ◽

pp. 930-939 ◽

Cited By ~ 49

Author(s):

SJ Szilvassy ◽

PM Lansdorp ◽

RK Humphries ◽

AC Eaves ◽

CJ Eaves

Keyword(s):

Simple Procedure ◽

Hematopoietic Cells ◽

Stem Cell Population ◽

Proliferative Potential ◽

Vitro Assay ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

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Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis

Circulation Research ◽

10.1161/res.113.suppl_1.a290 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Haruko Nakano ◽

Xiaoqian Liu ◽

Armin Arshi ◽

Ben van Handel ◽

Rajkumar Sasidharan ◽

...

Keyword(s):

De Novo ◽

Ex Vivo ◽

Embryonic Stem ◽

Circulatory System ◽

Lineage Tracing ◽

Mouse Heart ◽

Heart Tube ◽

Cardiac Progenitors ◽

Definitive Hematopoiesis

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

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Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽

10.1182/blood-2011-01-330514 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4773-4777 ◽

Cited By ~ 115

Author(s):

Hal E. Broxmeyer ◽

Man-Ryul Lee ◽

Giao Hangoc ◽

Scott Cooper ◽

Nutan Prasain ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

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