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Hematopoietic stem cells (HSCs) guarantee the continuous supply of all blood lineages during life. In response to stress, HSCs are capable of extensive proliferative expansion, whereas in steady state, HSCs largely remain in a quiescent state to prevent their exhaustion. DNA replication is a very complex process, where many factors need to exert their functions in a perfectly concerted manner. Mini-chromosome-maintenance protein 10 (Mcm10) is an important replication factor, required for proper assembly of the eukaryotic replication fork. In this report, we use zebrafish to study the role of mcm10 during embryonic development, and we show that mcm10 specifically regulates HSC emergence from the hemogenic endothelium. We demonstrate that mcm10-deficient embryos present an accumulation of DNA damages in nascent HSCs, inducing their apoptosis. This phenotype can be rescued by knocking down p53. Taken all together, our results show that mcm10 plays an important role in the emergence of definitive hematopoiesis., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

During development, hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium and will expand in a transient hematopoietic niche. Prostaglandin E 2 (PGE2) is essential during vertebrate development and HSC specification, but its precise source in the embryo remains elusive. Here, we show that in the zebrafish embryo, PGE2 synthesis genes are expressed by distinct stromal cell populations, myeloid (neutrophils, macrophages), and endothelial cells of the caudal hematopoietic tissue. Ablation of myeloid cells, which produce the PGE2 precursor prostaglandin H 2 (PGH2), results in loss of HSCs in the caudal hematopoietic tissue, which could be rescued by exogeneous PGE2 or PGH2 supplementation. Endothelial cells contribute by expressing the PGH2 import transporter slco2b1 and ptges3, the enzyme converting PGH2 into PGE2. Of note, differential niche cell expression of PGE2 biosynthesis enzymes is also observed in the mouse fetal liver. Taken altogether, our data suggest that the triad composed of neutrophils, macrophages, and endothelial cells sequentially and synergistically contributes to blood stem cell expansion during vertebrate development., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

During early vertebrate development, hematopoietic stem and progenitor cells (HSPCs) are produced in hemogenic endothelium located in the dorsal aorta, before they migrate to a transient niche where they expand to the fetal liver and the caudal hematopoietic tissue, in mammals and zebrafish, respectively. In zebrafish, previous studies have shown that the extracellular matrix (ECM) around the aorta must be degraded to enable HSPCs to leave the aortic floor and reach blood circulation. However, the role of the ECM components in HSPC specification has never been addressed. In this study, hapln1b, a key component of the ECM, was specifically expressed in hematopoietic sites in the zebrafish embryo. Gain- and loss-of-function experiments all resulted in the absence of HSPCs in the early embryo, showing that hapln1b is necessary, at the correct level, to specify HSPCs in the hemogenic endothelium. Furthermore, the expression of hapln1b was necessary to maintain the integrity of the ECM through its link domain. By combining functional analyses and computer modeling, we showed that kitlgb interacts with the ECM to specify HSPCs. The findings show that the ECM is an integral component of the microenvironment and mediates the cytokine signaling that is necessary for HSPC specification., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that Cx41.8 is important to lower ROS levels in HSPCs. We also demonstrate a new role for ifi30, known to be involved in the immune response. In the hematopoietic niche, Ifi30 can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver., (© 2021. The Author(s).)

In most vertebrates, the yolk sac (YS) represents the very first tissue where blood cells are detected. Therefore, it was thought for a long time that it generated all the blood cells present in the embryo. This model was challenged using different animal models, and we now know that YS hematopoietic precursors are mostly transient although their contribution to the adult system cannot be excluded. In this review, we aim at properly define the different waves of blood progenitors that are produced by the YS and address the fate of each of them. Indeed, in the last decade, many evidences have emphasized the role of the YS in the emergence of several myeloid tissue-resident adult subsets. We will focus on the development of microglia, the resident macrophages in the central nervous system, and try to untangle the recent controversy about their origin.

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 irf8 null 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., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Hematopoiesis maintains the entire blood system, and dysregulation of this process can lead to malignancies (leukemia), immunodeficiencies or red blood cell diseases (anemia, polycythemia vera). We took advantage of the zebrafish model that shares most of the genetic program involved in hematopoiesis with mammals to characterize a new gene of unknown function, si:ch73-299h12.2 , which is expressed in the erythroid lineage during primitive, definitive and adult hematopoiesis. This gene, required during primitive and definitive erythropoiesis, encodes a C2H2 zinc-finger protein that inhibits BMP signaling. We therefore named this gene blood-inducing factor 1 and BMP inhibitory factor 1 ( bif1 ). We identified a bif1 ortholog in Sinocyclocheilus rhinocerous , another fish, and in the mouse genome. Both genes also inhibit BMP signaling when overexpressed in zebrafish. In conclusion, we have deorphanized a new zebrafish gene of unknown function: bif1 codes for a zinc-finger protein that inhibits BMP signaling and also regulates primitive erythropoiesis and definitive hematopoiesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Rare hematopoietic stem cells (HSCs) can self-renew, establish the entire blood system and represent the basis of regenerative medicine applied to hematological disorders. Clinical use of HSCs is however limited by their inefficient expansion ex vivo , creating a need to further understand HSC expansion in vivo . After embryonic HSCs are born from the hemogenic endothelium, they migrate to the embryonic/fetal niche, where the future adult HSC pool is established by considerable expansion. This takes place at different anatomical sites and is controlled by numerous signals. HSCs then migrate to their adult niche, where they are maintained throughout adulthood. Exactly how HSC expansion is controlled during embryogenesis remains to be characterized and is an important step to improve the therapeutic use of HSCs. We will review the current knowledge of HSC expansion in the different fetal niches across several model organisms and highlight possible clinical applications.

Shear stress, a blood flow-induced frictional force, is essential in the control of endothelial cell (EC) homeostasis. High laminar shear stress (HLSS), as observed in straight parts of arteries, assures a quiescent non-activated endothelium through the induction of Krüppel-like transcription factors (KLFs). Connexin40 (Cx40)-mediated gap junctional communication is known to contribute to a healthy endothelium by propagating anti-inflammatory signals between ECs, however, the molecular basis of the transcriptional regulation of Cx40 as well as its downstream effectors remain poorly understood. Here, we show that flow-induced KLF4 regulated Cx40 expression in a mouse EC line. Chromatin immunoprecipitation in ECs revealed that KLF4 bound to three predicted KLF consensus binding sites in the Cx40 promoter. HLSS-dependent induction of Cx40 expression was confirmed in primary human ECs. The downstream effects of Cx40 modulation in ECs exposed to HLSS were elucidated by an unbiased transcriptomics approach. Cell cycle progression was identified as an important downstream target of Cx40 under HLSS. In agreement, an increase in the proportion of proliferating cell nuclear antigen (PCNA)-positive ECs and a decrease in the proportion of ECs in the G0/G1 phase were observed under HLSS after Cx40 silencing. Transfection of communication-incompetent HeLa cells with Cx40 demonstrated that the regulation of proliferation by Cx40 was not limited to ECs. Using a zebrafish model, we finally showed faster intersegmental vessel growth and branching into the dorsal longitudinal anastomotic vessel in embryos knock-out for the Cx40 orthologs Cx41.8 and Cx45.6 . Most significant effects were observed in embryos with a mutant Cx41.8 encoding for a channel with reduced gap junctional function. Faster intersegmental vessel growth in Cx41.8 mutant embryos was associated with increased EC proliferation as assessed by PH3 immunostaining. Our data shows a novel evolutionary-conserved role of flow-driven KLF4-dependent Cx40 expression in endothelial quiescence that may be relevant for the control of atherosclerosis and diseases involving sprouting angiogenesis.

Microglia, the tissue-resident macrophages of the CNS, represent major targets for therapeutic intervention in a wide variety of neurological disorders. Efficient reprogramming protocols to generate microglia-like cells in vitro using patient-derived induced pluripotent stem cells will, however, require a precise understanding of the cellular and molecular events that instruct microglial cell fates. This remains a challenge since the developmental origin of microglia during embryogenesis is controversial. Here, using genetic tracing in zebrafish, we uncover primitive macrophages as the unique source of embryonic microglia. We also demonstrate that this initial population is transient, with primitive microglia later replaced by definitive microglia that persist throughout adulthood. The adult wave originates from cmyb-dependent hematopoietic stem cells. Collectively, our work challenges the prevailing model establishing erythro-myeloid progenitors as the sole and direct microglial precursor and provides further support for the existence of multiple waves of microglia, which originate from distinct hematopoietic precursors., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding the molecular pathways controlling hematopoietic stem cell specification and expansion is a necessary milestone to perform regenerative medicine. Here, we used the zebrafish model to study the role of the ckit signaling pathway in this process. We show the importance of kitb/kitlgb signaling in the specification and expansion of hematopoietic stem cells (HSCs), in the hemogenic endothelium and caudal hematopoietic tissue (CHT), respectively. Moreover, we identified the zebrafish ortholog of Oncostatin M (osm) in the zebrafish genome. We show that the osm/osmr pathway acts upstream of kitb during specification of the hemogenic endothelium, while both pathways act synergistically to expand HSCs in the CHT. Moreover, we found that osm, in addition to its role in promoting HSC proliferation, inhibits HSC commitment to the lymphoid fate. Altogether, our data identified two cytokines, kitlgb and osm, secreted by the vascular niche, that control HSCs during early embryonic development., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1-ZBTB11-TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.

During hematopoiesis, the balance between proliferation, differentiation, and apoptosis is tightly regulated in order to maintain homeostasis. Failure in these processes can ultimately lead to uncontrolled proliferation and leukemia. Phosphatase and tensin homolog (PTEN) is one of the molecular pathways involved in this balance. By opposing PI3-kinases, PTEN inhibits proliferation and promotes differentiation and is thus considered a tumor suppressor. Indeed, PTEN is frequently mutated in many cancers, including leukemias. Loss of PTEN often leads to lymphoid cancers. However, little is known about the molecular events that regulate PTEN signaling during lymphopoiesis. In this study, we used zebrafish to address this. We report that N-myc downstream-regulated gene 1b (ndrg1b) rescues lymphoid differentiation after PTEN inhibition. We also show that a previously uncharacterized gene, fam49ab, inhibits T-cell differentiation, a phenotype that can be rescued by ndrg1b We propose that ndrg1b and fam49ab are 2 new modulators of PTEN signaling that control lymphoid differentiation in the zebrafish thymus., (© 2016 by The American Society of Hematology.)

In mammals, embryonic hematopoiesis occurs in successive waves, culminating with the emergence of hematopoietic stem cells (HSCs) in the aorta. HSCs first migrate to the fetal liver (FL), where they expand, before they seed the bone marrow niche, where they will sustain hematopoiesis throughout adulthood. In zebrafish, HSCs emerge from the dorsal aorta and colonize the caudal hematopoietic tissue (CHT). Recent studies showed that they interact with endothelial cells (ECs), where they expand, before they reach their ultimate niche, the kidney marrow. We identified tfec, a transcription factor from the mitf family, which is highly enriched in caudal endothelial cells (cECs) at the time of HSC colonization in the CHT. Gain-of-function assays indicate that tfec is capable of expanding HSC-derived hematopoiesis in a non-cell-autonomous fashion. Furthermore, tfec mutants (generated by CRISPR/Cas9) showed reduced hematopoiesis in the CHT, leading to anemia. Tfec mediates these changes by increasing the expression of several cytokines in cECs from the CHT niche. Among these, we found kitlgb, which could rescue the loss of HSCs observed in tfec mutants. We conclude that tfec plays an important role in the niche to expand hematopoietic progenitors through the modulation of several cytokines. The full comprehension of the mechanisms induced by tfec will represent an important milestone toward the expansion of HSCs for regenerative purposes., (© 2016 by The American Society of Hematology.)

The neurocranium generates most of the craniofacial skeleton and consists of prechordal and postchordal regions. Although development of the prechordal is well studied, little is known of the postchordal region. Here we characterize a signaling hierarchy necessary for postchordal neurocranial development involving Fibroblast growth factor (Fgf) signaling for early specification of mesodermally-derived progenitor cells. The expression of hyaluron synthetase 2 (has2) in the cephalic mesoderm requires Fgf signaling and Has2 function, in turn, is required for postchordal neurocranial development. While Hedgehog (Hh)-deficient embryos also lack a postchordal neurocranium, this appears primarily due to a later defect in chondrocyte differentiation. Inhibitor studies demonstrate that postchordal neurocranial development requires early Fgf and later Hh signaling. Collectively, our results provide a mechanistic understanding of early postchordal neurocranial development and demonstrate a hierarchy of signaling between Fgf and Hh in the development of this structure., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.

The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK) function.

The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

Recent studies have revealed that definitive hematopoiesis in vertebrates initiates through the formation of a non-self-renewing progenitor with limited multilineage differentiation potential termed the erythromyeloid progenitor (EMP). EMPs are specified before hematopoietic stem cells (HSCs), which self-renew and are capable of forming all mature adult blood lineages including lymphoid cells. Despite their differences, EMPs and HSCs share many phenotypic traits, making precise study of their respective functions difficult. Here, we examine whether embryonic specification of EMPs requires Notch signaling as has been shown for HSCs. In mindbomb mutants, which lack functional Notch ligands, we show that EMPs are specified normally: we detect no significant differences in cell number, gene expression, or differentiation capacity between EMPs purified from wild-type (WT) or mindbomb mutant embryos. Similarly N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a chemical inhibitor of Notch receptor activation, has no effect on EMP specification. These studies establish that HSCs are the only hematopoietic precursor that requires Notch signaling and help to clarify the signaling events underlying the specification of the 2 distinct waves of definitive hematopoiesis.

A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of haematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that haematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from haemogenic endothelial cells (ECs) lining the aortic floor. Additional in vitro studies have similarly indicated that the haematopoietic progeny of ESCs arise through intermediates with endothelial potential. Here we have used the unique strengths of the zebrafish embryo to image directly the generation of HSCs from the ventral wall of the dorsal aorta. Using combinations of fluorescent reporter transgenes, confocal time-lapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic haemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from haemogenic endothelium are the lineal founders of the adult haematopoietic system.

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.

Purpose of Review: A wealth of new experimental evidence has been published over the past year that has helped refine our models of blood cell development. We will review this information, discuss the current models of hematopoietic ontogeny and provide perspective on current and future research directions, with an emphasis on how studies in the zebrafish are helping us better understand how hematopoietic stem cells are formed in the vertebrate embryo., Recent Findings: Several important studies have been published recently addressing the embryonic development of hematopoietic stem cells. These studies have helped clarify several controversial topics in developmental hematopoiesis, including the concepts of the hemangioblast and hemogenic endothelium. In particular, the postulate that hematopoietic stem cells arise through hemogenic endothelial intermediates has been greatly strengthened by a collection of convincing publications reviewed below., Summary: A precise understanding of how hematopoietic stem cells are patterned during development has important implications for both developmental biology and regenerative medicine. Since hematopoietic stem cells are the only hematopoietic cells capable of lifelong, multilineage blood cell production, understanding the stepwise, molecular processes of their instruction from mesoderm is key to replicating these events in vitro from pluripotent embryonic stem cells.

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the "molar tooth sign" on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

For the formation of lymph nodes and Peyer's patches, lymphoid tissue inducer (LTi) cells are crucial in triggering stromal cells to recruit and retain hematopoietic cells. Although LTi cells have been observed in fetal spleen, not much is known about fetal spleen development and the role of LTi cells in this process. Here, we show that LTi cells collect in a periarteriolar manner in fetal spleen at the periphery of the white pulp anlagen. Expression of the homeostatic chemokines can be detected in stromal and endothelial cells, suggesting that LTi cells are attracted by these chemokines. As lymphotoxin (LT)alpha1beta2 can be detected on B cells but not LTi cells in neonatal spleen, starting at 4 days after birth, the earliest formation of the white pulp in fetal spleen occurs in a LTalpha1beta2-independent manner. The postnatal development of the splenic white pulp, involving the influx of T cells, depends on LTalpha1beta2 expressed by B cells.

Development of the vertebrate blood lineages is complex, with multiple waves of hematopoietic precursors arising in different embryonic locations. Monopotent, or primitive, precursors first give rise to embryonic macrophages or erythrocytes. Multipotent, or definitive, precursors are subsequently generated to produce the adult hematopoietic lineages. In both the zebrafish and the mouse, the first definitive precursors are committed erythromyeloid progenitors (EMPs) that lack lymphoid differentiation potential. We have previously shown that zebrafish EMPs arise in the posterior blood island independently from hematopoietic stem cells (HSCs). In this report, we demonstrate that a fourth wave of hematopoietic precursors arises slightly later in the zebrafish aorta/gonad/mesonephros (AGM) equivalent. We have identified and prospectively isolated these cells by CD41 (itga2b) and cmyb expression. Unlike EMPs, CD41(+) AGM cells colonize the thymus to generate rag2(+) T lymphocyte precursors. Timelapse imaging and lineage tracing analyses demonstrate that AGM-derived precursors use a previously undescribed migration pathway along the pronephric tubules to initiate adult hematopoiesis in the developing kidney, the teleostean equivalent of mammalian bone marrow. Finally, we have analyzed the gene expression profiles of EMPs and AGM precursors to better understand the molecular cues that pattern the first definitive hematopoietic cells in the embryo. Together, these studies suggest that expression of CD41 and cmyb marks nascent HSCs in the zebrafish AGM, and provide the means to further dissect HSC generation and function in the early vertebrate embryo.

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. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, 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 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. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.

The role of the fetal spleen in hematopoeisis remains largely unknown. In this particular environment, we show that hematopoietic stem cells do not proliferate, but that they lose multipotency and differentiate exclusively into mature macrophages. B lymphocytes in the spleen derive from committed B cell precursors that are likely to have immigrated from the fetal liver. We developed fetal spleen stromal cell lines that are unique in their capacity to expand myeloid precursors, resulting in large numbers of mature macrophages. These lines secrete high levels of anti-inflammatory molecules. By phenotype, fetal splenic macrophages are reminiscent of their adult counterparts found in the red pulp. We postulate that F4/80(+) splenic macrophages participate in fetal erythropoiesis, as well as in the formation of the splenic architecture.

The existence of macrophages (Mphi) of yolk-sac (YS) origin has been reported in all vertebrate models. However, the nature of their precursors and pathways of differentiation have not been elucidated. Phenotypic and differentiation potential analyses of YS at 7.5 to 10 postcoital days (dpc), performed in CX3CR1(GFP) embryos, allowed us to discern 3 independent Mphi populations. A first transient wave consisted of mature, maternal-derived Mphipresent as early as 7.5 to 8 dpc. A second wave of committed Mphi precursors arose at 8 dpc (2-4 somite stage) and was followed by a third wave of erythromyeloid precursors (4-6 somite stage). Both types of precursors displayed similar phenotypes and gave rise to CX3CR1/green fluorescent protein (GFP)-positive Mphi, but differed by their differentiation potential, at the clonal level. The combined data of phenotypic, gene-expression, and in situ analyses allowed us to conclude that the previously named "primitive Mphi" corresponded to a mixture of the first transient wave and committed Mphi precursors. Both YS-derived precursors followed a developmental pathway common to adult Mphi and could be qualified as definitive.

Little is known about hematopoietic stem cell (HSC) development from mesoderm. To gain more information on the intraembryonic HSC site of origin, we purified multipotent hematopoietic progenitors from the aorta-gonads-mesonephros (AGM) of mice. This population, expressing c-Kit, AA4.1, CD31, and CD41, but not Flk1, and mainly negative for CD45, proved capable of long-term reconstitution in sublethally irradiated Rag2gammac(-/-) recipients. We assigned the expression of GATA-2, GATA-3, and lmo2 to AGM-HSC, whereas erythromyeloid progenitors express only GATA-2. This unique combination of surface markers and transcription factors could be allocated in the AGM to the intraaortic clusters and the subaortic patches underlying aortic endothelial cells. Taken together, those data indicate that embryonic HSCs (i) differ from their fetal liver and adult counterpart by the low expression of CD45, (ii) do not colocalize with aortic endothelial cells as previously thought, and (iii) are localized, at 10.5 days postcoitum, in the splanchnic mesoderm underlying aortic endothelial cells, within GATA-3(+)CD31(+) cell clusters.

The progress of the last few years in the understanding of hematopoietic cell development during embryogenesis resulted from a combination of experimental approaches used in hematology and developmental biology. This methodology has been particularly powerful for the analysis of the earliest steps of hematopoietic ontogeny because it allows for the first time the demonstration of the existence of two independent sites of hematopoietic cell generation. Here, we describe the methods used in our laboratories to characterize the phenotype and differentiation potential of the primordial hematopoietic precursors as well as their localization in the mouse embryo. This multidisciplinary approach is required to explore the mechanisms of hematopoietic cell generation.

Finding ways to enhance remyelination is a major challenge in treating demyelinating diseases. Recent studies have suggested that circulating bone marrow cells can home in brain and transdifferentiate into neural cells. To ask whether hematopoietic precursors can form myelinating cells, we investigated the neuropoietic potential of embryonic precursors sorted from the mouse aorta-gonads-mesonephros (AGM) region. This cell fraction is capable of long-term hematopoietic reconstitution and generates colonies containing multipotential precursors and lymphoid or erythro-myeloid progenies. When cultured in hematopoietic growth conditions, a fraction of CD45-positive AGM cells coexpress neural markers such as nestin, the polysialylated form of neural cell adhesion molecule, the betaIII tubulin isoform, and glial fibrillary acidic protein. However, when hematopoietic precursors containing green fluorescent protein were cocultured with embryonic striatal precursors into neurospheres, they maintained their hematopoietic phenotype without undergoing differentiation into neurons, astrocytes, or oligodendrocytes. After intraventricular grafting, hematopoietic precursors integrated into the brain of wild-type or hypomyelinated newborn shiverer mice and gave rise to microglia but not neurons or glia. In contrast, when wild-type embryonic striatal neurospheres were grafted in shiverer, they formed numerous myelin internode patches. Even when neural and hematopoietic precursors were grafted together into shiverer mice, only neural precursors generated myelin-forming cells and synthesized myelin. Thus, embryonic neurospheres have myelin repair properties not shown by embryonic hematopoietic precursors. This suggests that the use of multipotential neural precursors to generate myelin-forming cells remains one of the most promising avenues toward remyelination therapies.